Methods of predicting toxicity

ABSTRACT

Described herein are compounds useful for the treatment and investigation of diseases, methods for the prediction of in vivo toxicity of compounds useful for the treatment and investigation of diseases, and methods of discovering and identifying compounds useful for the treatment and investigation of diseases that have reduced in vivo toxicity.

This application claims priority under U.S.C. 119(e) to U.S. ProvisionalApplication No. 61/565,835, filed Dec. 1, 2011, which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0101USSEQ.txt, created Dec. 3, 2012, which is 12 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND

Oligonucleotides have been used in various biological and biochemicalapplications. They have been used as primers and probes for thepolymerase chain reaction (PCR), as antisense agents used in targetvalidation, drug discovery and development, as ribozymes, as aptamers,and as general stimulators of the immune system. Antisense compoundshave been used to modulate target nucleic acids. Antisense compoundscomprising a variety of chemical modifications and motifs have beenreported. In certain instances, such compounds are useful as researchtools, diagnostic reagents, and as therapeutic agents. In certaininstances antisense compounds have been shown to modulate proteinexpression by binding to a target messenger RNA (mRNA) encoding theprotein. In certain instances, such binding of an antisense compound toits target mRNA results in cleavage of the mRNA. Antisense compoundsthat modulate processing of a pre-mRNA have also been reported. Suchantisense compounds alter splicing, interfere with polyadenlyation orprevent formation of the 5′-cap of a pre-mRNA. Certain antisensecompounds have undesired toxixity. See e.g., Swayze et al., “Antisenseoligonucleotides containing locked nucleic acid improve potency butcause significant hepatotoxicity in animals” Nucleic Acid Research(2007) 35(2):687-700.). This widespread use of antisense compounds andtheir vast potential as a potent therapeutic platform has led to anincreased demand for rapid, inexpensive, and efficient methods toanalyze and quantify the in vitro and in vivo properties of thesecompounds.

SUMMARY

The present disclosure provides the following non-limited numberedembodiments:

Embodiment 1

A method of predicting the in vivo toxicity of an oligomeric compound,wherein the method comprises:

-   -   contacting a cell in vitro with the oligomeric compound; and    -   measuring the modulation of the amount or activity of one or        more off-target genes.

Embodiment 2

The method of embodiment 1, wherein the oligomeric compound comprises agapmer oligonucleotide consisting of 10 to 30 linked nucleosides,wherein the gapmer oligonucleotide has a 5′ wing region positioned atthe 5′ end of a deoxynucleotide gap, and a 3′ wing region positioned atthe 3′ end of the deoxynucleotide gap.

Embodiment 3

The method of embodiment 2, wherein each of the wing regions is betweenabout 1 to about 7 nucleotides in length.

Embodiment 4

The method of embodiment 2, wherein each of the wing regions is betweenabout 1 to about 3 nucleotides in length.

Embodiment 5

The method of embodiment 2, wherein the deoxy gap region is betweenabout 7 to about 18 nucleotides in length.

Embodiment 6

The method of embodiment 2, wherein the deoxy gap region is betweenabout 11 to about 18 nucleotides in length.

Embodiment 7

The method of embodiment 2, wherein the deoxy gap region is betweenabout 7 to about 10 nucleotides in length.

Embodiment 8

The method of any of embodiments 1 to 7, wherein the oligomeric compoundcomprises at least one modified nucleoside.

Embodiment 9

The method of embodiment 8, wherein the modified nucleoside is a bicylicmodified nucleoside.

Embodiment 10

The method of embodiment 9, wherein the bicylic modified nucleoside isan LNA nucleoside.

Embodiment 11

The method embodiment 9, wherein the bicylic modified nucleoside is a4′-CH₂—O-2′ nucleoside.

Embodiment 12

The method embodiment 9, wherein the bicylic modified nucleoside is a4′-CH(CH₃)—O-2′ nucleoside.

Embodiment 13

The method of embodiment 8, wherein the modified nucleoside is a2′-modified nucleoside.

Embodiment 14

The method of embodiment 12, wherein the 2′-modified nucleoside issubstituted at the 2′ position with a substituted or unsubstituted—O-alkyl or substituted or unsubstituted —O-(2-acetylamide), wherein thenon-bicyclic 2′-modified nucleoside comprises a 2′-OCH₃, 2′-O(CH₂)₂OCH₃,or 2′-OCH₂C(O)—NR₁R₂, wherein R₁ and R₂ are independently hydrogen orsubstituted or unsubstituted alkyl or, in the alternative, are takentogether to make a heterocyclic moiety.

Embodiment 15

The method of embodiment 1, wherein the oligomeric compound comprises agapmer oligonucleotide consisting of 10 to 30 linked nucleosides whereinthe gapmer oligonucleotide has a 5′ wing region positioned at the 5′ endof a deoxynucleotide gap, and a 3′ wing region positioned at the 3′ endof the deoxynucleotide gap, wherein at least one nucleoside of at leastone of the wing regions is a 4′ to 2′ bicyclic nucleoside, and whereinat least one nucleoside of at least one of the wing regions is anon-bicyclic 2′-modified nucleoside.

Embodiment 16

The method of embodiment 15, wherein the 3′ wing of the oligomericcompound comprises at least one 4′ to 2′ bicyclic nucleoside.

Embodiment 17

The method of any of embodiments 15 to 16, wherein the 5′ wing of theoligomeric compound comprises at least one 4′ to 2′ bicyclic nucleoside.

Embodiment 18

The method of any of embodiments 15 to 16, wherein the 3′ wing of theoligomeric compound comprises at least one non-bicyclic 2′ modifiednucleoside.

Embodiment 19

The method of any of embodiments 15 to 18, wherein the 5′ wing of theoligomeric compound comprises at least one non-bicyclic 2′-modifiednucleoside.

Embodiment 20

The method of embodiment 15, wherein the 3′ wing of the oligomericcompound comprises at least three 4′ to 2′ bicyclic nucleosides.

Embodiment 21

The method of embodiment 15, wherein the 3′ wing of the oligomericcompound comprises at least three non-bicyclic 2′-modified nucleosides.

Embodiment 22

The method of embodiment 15, wherein the 5′ wing of the oligomericcompound comprises at least three 4′ to 2′ bicyclic nucleosides.

Embodiment 23

The method of embodiment 15, wherein the 5′ wing of the oligomericcompound comprises at least three non-bicyclic 2′-modified nucleosides.

Embodiment 24

The method of embodiment 15, wherein the 5′ wing of the oligomericcompound comprises at least three 4′ to 2′ bicyclic nucleosides, andwherein the 3′ wing of the oligomeric compound comprises at least threenon-bicyclic 2′-modified nucleosides.

Embodiment 25

The method of embodiment 15, wherein the 3′ wing of the oligomericcompound comprises at least three 4′ to 2′ bicyclic nucleosides, andwherein the 5′ wing of the oligomeric compound comprises at least threenon-bicyclic 2′-modified nucleosides.

Embodiment 26

The method of any of embodiments 1 to 25, wherein the non-bicyclic2′-modified nucleoside is substituted at the 2′ position with asubstituted or unsubstituted —O-alkyl or substituted or unsubstituted—O-(2-acetylamide), wherein the non-bicyclic 2′-modified nucleosidecomprises a 2′-OCH₃, 2′-O(CH₂)₂OCH₃, or 2′-OCH₂C(O)—NR₁R₂, wherein R₁and R₂ are independently hydrogen or substituted or unsubstituted alkylor, in the alternative, are taken together to make a heterocyclicmoiety.

Embodiment 27

The method of embodiment 26, wherein the non-bicyclic 2′-modifiednucleoside is a 2′-O-methyl nucleoside.

Embodiment 28

The method of embodiment 26, wherein the non-bicyclic 2′-modifiednucleoside is a 2′-O(CH₂)₂OCH₃.

Embodiment 29

The method of any of embodiments 1 to 28, wherein the oligomericcompound comprises at least one modified internucleoside linkage.

Embodiment 30

The method of any of embodiments 1 to 29, wherein at least one modifiedinternucleoside linkage is a phosphorothioate linkage.

Embodiment 31

The method of any of embodiments 1 to 30, wherein the oligomericcompound comprises at least 3 phosphorothioate linkages.

Embodiment 32

The method of any of embodiments 1 to 31, wherein each internucleosidelinkage in the oligomeric compound comprises a phosphorothioate linkage.

Embodiment 33

The oligomeric compound of any of embodiments 1 to 32, wherein each ofthe wing regions is between about 1 to about 7 nucleosides in length.

Embodiment 34

The oligomeric compound of any of embodiments 1 to 32, wherein each ofthe wing regions is between about 1 to about 3 nucleosides in length.

Embodiment 35

The method of any of embodiments 1 to 34, wherein the method ofmeasuring modulation of the amount or activity of one or more off-targetgenes comprises measuring the increase in expression of one or moreoff-target genes and the reduction in expression of one or moreoff-target genes.

Embodiment 36

The method of any of embodiments 1 to 34, wherein the method ofmeasuring modulation of the amount or activity of one or more off-targetgenes comprises measuring the increase in expression of one or moreoff-target genes.

Embodiment 37

The method of embodiment 1, wherein the method of measuring modulationof the amount or activity of one or more off-target genes comprisesmeasuring the decrease in expression of one or more off-target genes.

Embodiment 38

The method of any of embodiments 1 to 37, wherein the off-target gene isa sentinel gene.

Embodiment 39

The method of any of embodiments 1 to 38, wherein at least one sentinelgene is selected from the group consisting of Fbx117, Fto, Gphn, Cadps2,Bcas3, Msi2, BC057079, Chn2, Tbc1d22a, Macrod1, Iqgap2, Vps13b, Atg10,Fggy, Odz3, Vps53, Cgn11, RAPTOR, Ptprk, Vti1a, Ubac2, Fars2, Ppm11,Adk, 0610012H03Rik, Itpr2, Sec1512///Exoc6b, Atp9b, Atxn1, Adcy9, Mcph1,Ppp3ca, Bre, Dus41, Rassf1, Mdm2, Brp16, 0610010K14Rik, Rce1, I1f2,Setd1a, and Gar1.

Embodiment 40

The method of any of embodiments 1 to 38, wherein at least two sentinelgenes are selected from the group consisting of Fbx117, Fto, Gphn,Cadps2, Bcas3, Msi2, BC057079, Chn2, Tbc1d22a, Macrod1, Iqgap2, Vps13b,Atg10, Fggy, Odz3, Vps53, Cgn11, RAPTOR, Ptprk, Vti1a, Ubac2, Fars2,Ppm11, Adk, 0610012H03Rik, Itpr2, Sec1512///Exoc6b, Atp9b, Atxn1, Adcy9,Mcph1, Ppp3ca, Bre, Dus41, Rassf1, Mdm2, Brp16, 0610010K14Rik, Rce1,I1f2, Setd1a, and Gar1.

Embodiment 41

The method of any of embodiments 1 to 38, wherein at least threesentinel genes are selected from the group consisting of Fbx117, Fto,Gphn, Cadps2, Bcas3, Msi2, BC057079, Chn2, Tbc1d22a, Macrod1, Iqgap2,Vps13b, Atg10, Fggy, Odz3, Vps53, Cgn11, RAPTOR, Ptprk, Vti1a, Ubac2,Fars2, Ppm11, Adk, 0610012H03Rik, Itpr2, Sec1512///Exoc6b, Atp9b, Atxn1,Adcy9, Mcph1, Ppp3ca, Bre, Dus41, Rassf1, Mdm2, Brp16, 0610010K14Rik,Rce1, I1f2, Setd1a, and Gar1.

Embodiment 42

The method of any of embodiments 1 to 38, wherein at least four sentinelgenes are selected from the group consisting of Fbx117, Fto, Gphn,Cadps2, Bcas3, Msi2, BC057079, Chn2, Tbc1d22a, Macrod1, Iqgap2, Vps13b,Atg10, Fggy, Odz3, Vps53, Cgn11, RAPTOR, Ptprk, Vti1a, Ubac2, Fars2,Ppm11, Adk, 0610012H03Rik, Itpr2, Sec1512///Exoc6b, Atp9b, Atxn1, Adcy9,Mcph1, Ppp3ca, Bre, Dus41, Rassf1, Mdm2, Brp16, 0610010K14Rik, Rce1,I1f2, Setd1a, and Gar1.

Embodiment 43

The method of any of embodiments 1 to 42, wherein one sentinel gene isFbx117.

Embodiment 44

The method of any of embodiments 1 to 43, wherein one sentinel gene isFto.

Embodiment 45

The method of any of embodiments 1 to 44, wherein one sentinel gene isGphn.

Embodiment 46

The method of any of embodiments 1 to 45, wherein one sentinel gene isCadps2.

Embodiment 47

The method of any of embodiments 1 to 46, wherein one sentinel gene isBcas3.

Embodiment 48

The method of any of embodiments 1 to 47, wherein one sentinel gene isMsi2.

Embodiment 49

The method of any of embodiments 1 to 48, wherein one sentinel gene isBC057079.

Embodiment 50

The method of any of embodiments 1 to 49, wherein one sentinel gene isChn2.

Embodiment 51

The method of any of embodiments 1 to 50, wherein one sentinel gene isTbc1d22a.

Embodiment 52

The method of any of embodiments 1 to 51, wherein one sentinel gene isMacrod1.

Embodiment 53

The method of any of embodiments 1 to 52, wherein one sentinel gene isIqgap2.

Embodiment 54

The method of any of embodiments 1 to 53, wherein one sentinel gene isVps13b.

Embodiment 55

The method of any of embodiments 1 to 54, wherein one sentinel gene isAtg10.

Embodiment 56

The method of any of embodiments 1 to 55, wherein one sentinel gene isFggy.

Embodiment 57

The method of any of embodiments 1 to 56, wherein one sentinel gene isOdz3.

Embodiment 58

The method of any of embodiments 1 to 57, wherein one sentinel gene isVps53.

Embodiment 59

The method of any of embodiments 1 to 58, wherein one sentinel gene isCgn11.

Embodiment 60

The method of any of embodiments 1 to 59, wherein one sentinel gene isRAPTOR.

Embodiment 61

The method of any of embodiments 1 to 60, wherein one sentinel gene isPtprk.

Embodiment 62

The method of any of embodiments 1 to 61, wherein one sentinel gene isVti1a.

Embodiment 63

The method of any of embodiments 1 to 62, wherein one sentinel gene isUbac2.

Embodiment 64

The method of any of embodiments 1 to 63, wherein one sentinel gene isFars2.

Embodiment 65

The method of any of embodiments 1 to 64, wherein one sentinel gene isPpm11.

Embodiment 66

The method of any of embodiments 1 to 65, wherein one sentinel gene isAdk.

Embodiment 67

The method of any of embodiments 1 to 66, wherein one sentinel gene is0610012H03Rik.

Embodiment 68

The method of any of embodiments 1 to 67, wherein one sentinel gene isItpr2.

Embodiment 69

The method of any of embodiments 1 to 68, wherein one sentinel gene isSec1512///Exoc6b.

Embodiment 70

The method of any of embodiments 1 to 69, wherein one sentinel gene isAtp9b.

Embodiment 71

The method of any of embodiments 1 to 70, wherein one sentinel gene isAtxn1.

Embodiment 72

The method of any of embodiments 1 to 71, wherein one sentinel gene isAdcy9.

Embodiment 73

The method of any of embodiments 1 to 72, wherein one sentinel gene isMcph1.

Embodiment 74

The method of any of embodiments 1 to 73, wherein one sentinel gene isPpp3ca.

Embodiment 75

The method of any of embodiments 1 to 74, wherein one sentinel gene isBre.

Embodiment 76

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of Adcy9, Ptprk, Tbc1d22a, and Exoc6b is measured.

Embodiment 77

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of Fbx117, Fto, Gphn, and Cadps2 is measured.

Embodiment 78

The method of any of embodiments 1 to 38, wherein the modulation of theincrease in expression of one or more of Dus41, Rassf1, Mdm2, Brp16,0610010K14Rik, Rce1, I1f2, Setd1a, and Gar1 is measured.

Embodiment 79

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of one or more of ADK, FTO, IQGAP2, PPP3CA, PTPRK,and/or RAPTOR is measured.

Embodiment 80

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of ADK and one or more of FTO, IQGAP2, PPP3CA, PTPRK,and/or RAPTOR is measured.

Embodiment 81

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of FTO and one or more of ADK, IQGAP2, PPP3CA, PTPRK,and/or RAPTOR is measured.

Embodiment 82

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of IQGAP2 and one or more of ADK, FTO, PPP3CA, PTPRK,and/or RAPTOR is measured.

Embodiment 83

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of PPP3CA and one or more of ADK, FTO, IQGAP2, PTPRK,and/or RAPTOR is measured.

Embodiment 84

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of PPP3CA and one or more of ADK, FTO, IQGAP2, PTPRK,and/or RAPTOR is measured.

Embodiment 85

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of PTPRK and one or more of ADK, FTO, IQGAP2, PPP3CA,and/or RAPTOR is measured.

Embodiment 86

The method of any of embodiments 1 to 38, wherein the modulation of theamount or activity of RAPTOR and one or more of ADK, FTO, IQGAP2,PPP3CA, and/or PTPRK is measured.

Embodiment 87

The method of any of embodiments 1 to 38, wherein the down-regulatedsentinel gene has a pre-mRNA length of greater than 176442 nucelobases.

Embodiment 88

The method of any of embodiments 1 to 38, wherein the down-regulatedsentinel gene has a pre-mRNA length of greater than 19862 nucleobases.

Embodiment 89

The method of any of embodiments 1 to 38, wherein the up-regulatedsentinel gene has a pre-mRNA length of less than 19862 nucleobases.

Embodiment 90

The method of any of embodiments 1 to 38, wherein the up-regulatedsentinel gene has a pre-mRNA length of less than 7673 nucleobases.

Embodiment 91

The method of any of embodiments 1 to 38, wherein the down-regulatedsentinel gene has an mRNA length of greater than 3962 nucelobases.

Embodiment 92

The method of any of embodiments 1 to 38, wherein the down-regulatedsentinel gene has an mRNA length of greater than 2652 nucleobases.

Embodiment 93

The method of any of embodiments 1 to 38, wherein the up-regulatedsentinel gene has an mRNA length of less than 2652 nucleobases.

Embodiment 94

The method of any of embodiments 1 to 38, wherein the up-regulatedsentinel gene has an mRNA length of less than 1879 nucleobases.

Embodiment 95

The method of any of embodiments 1 to 94, wherein the predicted in vivotoxicity of the oligomeric compound is predicted by measurement ofhepatotoxicity.

Embodiment 96

The method of any of embodiments 1 to 94, wherein the predicted in vivotoxicity of the oligomeric compound is predicted by a change in theamount of a liver enzyme.

Embodiment 97

The method of any of embodiments 1 to 94, wherein the predicted in vivotoxicity of the oligomeric compound is predicted by measurement of ALT.

Embodiment 98

The method of any of embodiments 1 to 94, wherein the predicted in vivotoxicity of the oligomeric compound is predicted by measurement of AST.

Embodiment 99

The method of any of embodiments 1 to 94, wherein the cell contactedwith the oligomeric compound in vitro is a bEnd3 cell.

Embodiment 100

An oligomeric compound identified by the method of any of embodiments 1to 99.

Embodiment 101

A method of administering the compound of embodiment 100 to an animal.

Embodiment 102

The in vitro method of determining the in vivo toxicity of any ofembodiments 1 to 100, wherein the method comprises administering theoligomeric compound to an animal.

Embodiment 103

A method of determining the in vivo toxicity of an oligomeric compound,wherein the method comprises:

-   -   contacting a cell with the oligomeric compound in vitro;    -   measuring modulation of the amount or activity of one or more        off-target genes;    -   determining the in vivo toxicity of the oligomeric compound        based on the level of amount or activity of the off-target        genes; and    -   administering the oligomeric compound to an animal.

Embodiment 104

The method of embodiment 103, wherein the off-target gene is a sentinelgene.

Embodiment 105

A method of predicting the in vivo or in vitro toxicity of an oligomericcompound, wherein the method comprises:

-   -   setting a minimum amount of complementarity between the        nucleobase sequence of the oligomeric compound and an off-target        gene;    -   determining the amount of complementarity between the sequence        of the oligomeric compound and a group of one or more off-target        genes in a genome;    -   setting a minimum number of off-target genes that have an equal        to or greater amount of complementarity between the sequence of        the oligomeric compound and a group of one or more off-target        genes; and    -   determining the number of off-target genes in a genome that have        an equal to or greater amount of complementarity between the        sequence of the oligomeric compound and a group of one or more        off-target genes.

Embodiment 106

The method of embodiment 105, wherein a computer is used to determinethe amount of complementarity between the sequence of the oligomericcompound and a group of one or more off-target genes.

Embodiment 107

The method of any one of embodiments 105 to 106, wherein a computer isused to determine the number of off-target genes that have an equal toor greater amount of complementarity between the sequence of theoligomeric compound and a group of one or more off-target genes.

Embodiment 108

The method of any one of embodiments 105 to 107, wherein the amount ofcomplementarity is a measure of the number of consecutive complementarynucleobases between the oligomeric compound and a group of one or moreoff-target genes.

Embodiment 109

The method of any one of embodiments 105 to 108, wherein each off-targetgene is a sentinel gene.

Embodiment 110

A method of identifying a sentinel gene, wherein the method comprises:

-   -   administering a compound to an animal;    -   assessing the toxicity of the compound at a timepoint after        administration of the compound; measuring the degree of        modulation of one or more one off-target genes;    -   calculating the correlation between the degree of off-target        gene modulation and toxicity;    -   identifying any off-target genes having a coefficient of        determination greater than 0.

Embodiment 111

The method of embodiment 110, wherein the coefficient of determinationis greater than 0.5.

Embodiment 112

The method of embodiment 110, wherein the coefficient of determinationis greater than 0.6.

Embodiment 113

The method of embodiment 110, wherein the coefficient of determinationis greater than 0.7.

Embodiment 114

The method of embodiment 110, wherein the coefficient of determinationis greater than 0.8.

Embodiment 115

The method of embodiment 110, wherein the coefficient of determinationis greater than 0.9.

Embodiment 116

The method of embodiment any of embodiments 110 to 115, wherein thetoxicity is assessed 24 hours after administration of the compound.

Embodiment 117

The method of any of embodiments 110 to 115, wherein the toxicity isassessed 48 hours after administration of the compound.

Embodiment 118

The method of any of embodiments 110 to 116, wherein the degree ofmodulation of one or more one off-target genes is greater than one-fold.

Embodiment 119

The method of any of embodiments 110 to 116, wherein the degree ofmodulation of one or more one off-target genes is greater than two-fold.

Embodiment 120

A method of predicting in vivo toxicity of an oligonucleotide comprising

-   -   comparing the nucleobase sequence of the oligonucleotide to the        nucleobase sequence of at least one sentinel gene transcript;

determining whether the oligonucleotide is complementary to any regionsof the at least one sentinel gene transcript;

predicting whether the oligonucleotide will hybridize to the sentinelgene transcript under physiologically relevant conditions; and

-   -   predicting toxicity based on the prediction of hybridization.

Embodiment 121

A method of identifying at least one antisense compound that ispredicted not to be toxic in vivo comprising:

-   -   identifying a set of potential antisense compounds, each having        a nuclebase sequence complementary to a target nucleic acid;    -   comparing the nucleobase sequence of each potential antisense        compound to the nucleobase sequence of at least one sentinel        gene transcript;    -   identifying potential antisense compounds having a nucleobase        sequence complementary to at least one sentinel gene transcript        as predicted toxic antisense compounds;    -   removing the predicted toxic compounds from the set of potential        antisense compounds;    -   identifying one or more of the remaining potential antisense        compounds as predicted not to be toxic in vivo.

Embodiment 122

The method of embodiment 120 or 121, wherein the predicted toxiccompounds are 100% complementary to at least one sentinel genetranscript.

Embodiment 123

The method of embodiment 122, wherein the predicted toxic compounds havenot more than one mismatch relative to at least one sentinel genetranscript.

Embodiment 124

The method of embodiment 122, wherein the predicted toxic compounds havenot more than two mismatches relative to at least one sentinel genetranscript.

Embodiment 125

The method of embodiment 120 or 121, wherein each potential antisensecompound is compared to the nucleobase sequence of at least two sentinelgene transcripts.

Embodiment 126

The method of embodiment 120 or 121, wherein each potential antisensecompound is compared to the nucleobase sequence of at least threesentinel gene transcripts.

Embodiment 127

The method of any of embodiments 120 to 126, wherein at least onesentinel gene is selected from the group consisting of Fbx117, Fto,Gphn, Cadps2, Bcas3, Msi2, BC057079, Chn2, Tbc1d22a, Macrod1, Iqgap2,Vps13b, Atg10, Fggy, Odz3, Vps53, Cgn11, RAPTOR, Ptprk, Vti1a, Ubac2,Fars2, Ppm11, Adk, 0610012H03Rik, Itpr2, Sec1512///Exoc6b, Atp9b, Atxn1,Adcy9, Mcph1, Ppp3ca, Bre, Dus41, Rassf1, Mdm2, Brp16, 0610010K14Rik,Rce1, I1f2, Setd1a, and Gar1.

Embodiment 128

The method of any of embodiments 1 to 127 comprising making at least oneantisense compound that is predicted not to be toxic in vivo and testingit in an animal.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

A. DEFINITIONS

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21′ edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Chemicalmodifications of oligonucleotides include nucleoside modifications(including sugar moiety modifications and nucleobase modifications) andinternucleoside linkage modifications. In reference to anoligonucleotide, chemical modification does not include differences onlyin nucleobase sequence.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moietyor a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is nota naturally occurring sugar moiety. Substituted sugar moieties include,but are not limited to furanosyls comprising substituents at the2′-position, the 3′-position, the 5′-position and/or the 4′-position.Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “2′-F nucleoside” refers to a nucleoside comprising asugar comprising fluoroine at the 2′ position. Unless otherwiseindicated, the fluorine in a 2′-F nucleoside is in the ribo position(replacing the OH of a natural ribose).

As used herein, “2′-(ara)-F” refers to a 2′-F substituted nucleoside,wherein the fluoro group is in the arabino position.

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside sub-units are capable of linking together and/or linking toother nucleosides to form an oligomeric compound which is capable ofhybridizing to a complementary oligomeric compound. Such structuresinclude rings comprising a different number of atoms than furanosyl(e.g., 4, 6, or 7-membered rings); replacement of the oxygen of afuranosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); orboth a change in the number of atoms and a replacement of the oxygen.Such structures may also comprise substitutions corresponding to thosedescribed for substituted sugar moieties (e.g., 6-membered carbocyclicbicyclic sugar surrogates optionally comprising additionalsubstituents). Sugar surrogates also include more complex sugarreplacements (e.g., the non-ring systems of peptide nucleic acid). Sugarsurrogates include without limitation morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “RNA-like nucleoside” means a modified nucleoside thatadopts a northern configuration and functions like RNA when incorporatedinto an oligonucleotide. RNA-like nucleosides include, but are notlimited to 3′-endo furanosyl nucleosides and RNA surrogates.

As used herein, “3′-endo-furanosyl nucleoside” means an RNA-likenucleoside that comprises a substituted sugar moiety that has a 3′-endoconformation. 3′-endo-furanosyl nucleosides include, but are not limitedto: 2′-MOE, 2′-F, 2′-OMe, LNA, ENA, and cEt nucleosides.

As used herein, “RNA-surrogate nucleoside” means an RNA-like nucleosidethat does not comprise a furanosyl. RNA-surrogate nucleosides include,but are not limited to hexitols and cyclopentanes.

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to anoligonucleotide or oligomeric compound. In general, conjugate groupsmodify one or more properties of the compound to which they areattached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group ofatoms used to attach a conjugate to an oligonucleotide or oligomericcompound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measureable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenlyation, addition of 5′-cap), and translation.

As used herein, “modulation” means a change of amount, activity, orquality when compared to the amount, activity, or quality prior tomodulation. For example, “modulation” of a nucleic acid includes anychange in the amount or activity of the nucleic acid. In certainembodiments, modulation of a nucleic acid is assessed by comparing theamount and/or activity of the nucleic acid in a sample before and afteran intervention or by comparing the amount and/or activity in one sampleto the amount or activity of the same gene in another sample. In certainembodiments, modulation of a nucleic acid includes, but is not limitedto, a change in the amount in which expression of a certain gene in onesample is reduced (e.g. down regulated) relative to expression of thesame gene in another sample. In certain embodiments, a decrease in theexpression (e.g. down regulation) of a gene describes a gene which hasbeen observed to have lower expression (e.g. lower mRNA levels), in onesample compared to another sample (e.g. a control). In certainembodiments, modulation of expression includes, but is not limited to,the amount in which expression of a certain gene in one sample isincreased (e.g. up regulated) relative to expression of the same gene inanother sample. In certain embodiments, an increase in the expression(up regulation) of a gene describes a gene which has been observed tohave higher expression (e.g. higher mRNA levels), in one sample comparedto another sample (e.g. a control).

As used herein, “activity” means performance of a function. In certainembodiments, activity of a nucleic acid includes, but is not limited to,expression of an encoded protein, modulation of expression of one ormore other nucleic acids, structural functions, and any other biologicalactivity performed by a nucleic acid.

As used herein, “amount” means amount or concentration.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound hybridizes, resulting in a desired antisenseactivity.

As used herein, “off-target nucleic acid” means a nucleic acid moleculeother than the target nucleic acid. Because some off-target nucleicacids may share some sequence homology with a target nucleic acid, incertain instances an antisense compound may hybridize to an off-targetnucleic acid. In certain embodiments, the amount, activity, orexpression of an off-target nucleic acid may be modulated by anantisense compound. Such modulation may have no consequences or mayresult in one or more antisense activity, including but not limited totoxicity. In certain embodiments, off-target nucleic acids include, butare not limited to, off-target genes.

As used herein, “sentinel gene” means a gene, the modulation of theamount or activity of which in vitro correlates with toxicity in vivo.In certain embodiments, toxicity is hepatotoxicity. In certainembodiments, sentinel genes include, but are not limited to, off-targetgenes. In certain embodiments, a decrease in expression of a sentinelgene in vitro correlates with an increase in AST levels in vivo. Incertain embodiments, a decrease in expression of a sentinel gene invitro correlates with an increase in ALT levels in vivo. In certainembodiments, an increase in expression of a sentinel gene in vitrocorrelates with toxicity in vivo. In certain embodiments, modulation ofthe amount or activity of a sentinel gene in vitro correlates with invivo toxicity with a coefficient of determination of at least 0.5. Incertain embodiments, modulation of the amount or activity of a sentinelgene in vitro correlates with in vivo toxicity with a coefficient ofdetermination of at least 0.6. In certain embodiments, modulation of theamount or activity of a sentinel gene in vitro correlates with in vivotoxicity with a coefficient of determination of at least 0.7. In certainembodiments, modulation of the amount or activity of a sentinel gene invitro correlates with in vivo toxicity with a coefficient ofdetermination of at least 0.8.

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not beenfully processed into mRNA. Pre-RNA includes one or more intron.

As used herein, “object RNA” means an RNA molecule other than a targetRNA, the amount, activity, splicing, and/or function of which ismodulated, either directly or indirectly, by a target nucleic acid. Incertain embodiments, a target nucleic acid modulates splicing of anobject RNA. In certain such embodiments, an antisense compound modulatesthe amount or activity of the target nucleic acid, resulting in a changein the splicing of an object RNA and ultimately resulting in a change inthe activity or function of the object RNA.

As used herein, “microRNA” means a naturally occurring, small,non-coding RNA that represses gene expression of at least one mRNA. Incertain embodiments, a microRNA represses gene expression by binding toa target site within a 3′ untranslated region of an mRNA. In certainembodiments, a microRNA has a nucleobase sequence as set forth inmiRBase, a database of published microRNA sequences found athttp://microrna.sanger.ac.uk/sequences/. In certain embodiments, amicroRNA has a nucleobase sequence as set forth in miRBase version 18released November 2011, which is herein incorporated by reference in itsentirety.

As used herein, “microRNA mimic” means an oligomeric compound having asequence that is at least partially identical to that of a microRNA. Incertain embodiments, a microRNA mimic comprises the microRNA seed regionof a microRNA. In certain embodiments, a microRNA mimic modulatestranslation of more than one target nucleic acids. In certainembodiments, a microRNA mimic is double-stranded.

As used herein, “differentiating nucleobase” means a nucleobase thatdiffers between two nucleic acids. In certain instances, a target regionof a target nucleic acid differs by 1-4 nucleobases from a non-targetnucleic acid. Each of those differences is referred to as adifferentiating nucleobase. In certain instances, a differentiatingnucleobase is a single-nucleotide polymorphism.

As used herein, “target-selective nucleoside” means a nucleoside of anantisense compound that corresponds to a differentiating nucleobase of atarget nucleic acid.

As used herein, “allele” means one of a pair of copies of a geneexisting at a particular locus or marker on a specific chromosome, orone member of a pair of nucleobases existing at a particular locus ormarker on a specific chromosome, or one member of a pair of nucleobasesequences existing at a particular locus or marker on a specificchromosome. For a diploid organism or cell or for autosomal chromosomes,each allelic pair will normally occupy corresponding positions (loci) ona pair of homologous chromosomes, one inherited from the mother and oneinherited from the father. If these alleles are identical, the organismor cell is said to be “homozygous” for that allele; if they differ, theorganism or cell is said to be “heterozygous” for that allele.“Wild-type allele” refers to the genotype typically not associated withdisease or dysfunction of the gene product. “Mutant allele” refers tothe genotype associated with disease or dysfunction of the gene product.

As used herein, “targeting” or “targeted to” means the association of anantisense compound to a particular target nucleic acid molecule or aparticular region of a target nucleic acid molecule. An antisensecompound targets a target nucleic acid if it is sufficientlycomplementary to the target nucleic acid to allow hybridization underphysiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity under stringent conditions. Complementary oligomericcompounds need not have nucleobase complementarity at each nucleoside.Rather, some mismatches are tolerated. In certain embodiments,complementary oligomeric compounds or regions are complementary at 70%of the nucleobases (70% complementary). In certain embodiments,complementary oligomeric compounds or regions are 80% complementary. Incertain embodiments, complementary oligomeric compounds or regions are90% complementary. In certain embodiments, complementary oligomericcompounds or regions are 95% complementary. In certain embodiments,complementary oligomeric compounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomericcompound that is not capable of pairing with a nucleobase at acorresponding position of a second oligomeric compound, when the firstand second oligomeric compound are aligned. Either or both of the firstand second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site. In certainembodiments, an antisense oligonucleotide specifically hybridizes tomore than one target site.

As used herein, “fully complementary” in reference to an oligonucleotideor portion thereof means that each nucleobase of the oligonucleotide orportion thereof is capable of pairing with a nucleobase of acomplementary nucleic acid or contiguous portion thereof. Thus, a fullycomplementary region comprises no mismatches or unhybridized nucleobasesin either strand.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modification motif” means a pattern of chemicalmodifications in an oligomeric compound or a region thereof. Motifs maybe defined by modifications at certain nucleosides and/or at certainlinking groups of an oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligomeric compound or a region thereof. Thelinkages of such an oligomeric compound may be modified or unmodified.Unless otherwise indicated, motifs herein describing only nucleosidesare intended to be nucleoside motifs. Thus, in such instances, thelinkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligomeric compound or region thereof. The nucleosides of such anoligomeric compound may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleoside have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substuent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentinvention have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms differs from the atomor a group of atoms normally present in the named functional group. Incertain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino(═NR_(bb)), amido (—C(O)N(R_(bb))(R_(bb)) or—N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))—(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)).Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl. Selected substituentswithin the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

B. METHODS OF PREDICTING IN VIVO TOXICITY

Provided herein are methods for determining the in vitro and in vivotoxicity of oligomeric compounds. In certain embodiments, the methodsgenerally comprise contacting a cell with an oligomeric compound invitro, measuring the modulation of the activity or amount of one or moreoff-target genes and predicting the in vivo toxicity of the oligomericcompound based on the in vitro modulation of the activity or amount ofone or more of the off-target genes. In certain embodiments, the generalmethods disclosed herein will enable one having skill in the art torapidly screen large numbers of new or previously known oligomericcompounds in vitro and predict whether such test oligomeric compoundswill be toxic in vivo, based on the in vitro modulation of the amount oractivity of certain off-target genes. Thus, the time and expense ofadministering numerous oligomeric compounds to animals to determine invivo toxicity may be reduced, and one may more rapidly identify andavoid oligomeric compounds that may have potentially toxic in vivoproperties.

In certain embodiments, the method generally comprises identifying oneor more off-target genes, the up- or down-regulation of which in vitrocorrelates with an increase in toxicity in vivo. In certain embodiments,once such off-target genes are identified, the invention providesmethods of screening oligomeric compounds in vitro to determine whetherthey up- or down-regulate such off-target genes. In certain embodiments,the methods disclosed herein enable one having skill in the art toaccurately predict the in vivo toxicity of a given oligomeric compoundthrough the in vitro measurement of certain down-regulated off-targetgenes. In certain embodiments, the methods disclosed herein enable onehaving skill in the art to accurately predict the in vivo toxicity of agiven oligomeric compound through the in vitro measurement of certainup-regulated off-target genes.

a. Toxicity

In vitro or in vivo toxicity may be measured by any method known tothose having skill in the art. In some embodiments, toxicity is measuredby liver activity. In some embodiments, toxicity is measured by kidneyactivity. In some embodiments, toxicity is measured by pancreasactivity. In some embodiments, toxicity is measured by assessingcirculating liver enzymes such as Aspartate transaminase (AST) and/orAlanine transaminase (ALT). In certain such embodiments, AST and/or ALTlevels at timepoints after the administration of a test oligomericcompound are compared to baseline values obtained prior toadministration, to those of control animals that did not receive testoligomeric compound, or to values known to be associated with normalanimals from previous experiments (historical controls) or fromliterature. In certain embodiments, toxicity is measured by assessingalkaline phosphatase (ALP) levels. In certain such embodiments, ALPlevels at timepoints after the administration of a test oligomericcompound are compared to baseline values obtained prior toadministration, to those of control animals that did not receive testoligomeric compound, or to values known to be associated with normalanimals from previous experiments (historical controls) or fromliterature. In certain embodiments, toxicity is measured by assessingtotal bilirubin (TBIL) levels. In certain such embodiments, TBIL levelsat timepoints after the administration of a test oligomeric compound arecompared to baseline values obtained prior to administration, to thoseof control animals that did not receive test oligomeric compound, or tovalues known to be associated with normal animals from previousexperiments (historical controls) or from literature. In certainembodiments, toxicity is measured by assessing albumin levels. Incertain such embodiments, albumin levels at timepoints after theadministration of a test oligomeric compound are compared to baselinevalues obtained prior to administration, to those of control animalsthat did not receive test oligomeric compound, or to values known to beassociated with normal animals from previous experiments (historicalcontrols) or from literature. In certain embodiments, toxicity ismeasured by assessing serum glucose levels. In certain such embodiments,serum glucose levels at timepoints after the administration of a testoligomeric compound are compared to baseline values obtained prior toadministration, to those of control animals that did not receive testoligomeric compound, or to values known to be associated with normalanimals from previous experiments (historical controls) or fromliterature. In certain embodiments, toxicity is measured by assessinglactate dehydrogenase (LDH) levels. In certain such embodiments, lactatedehydrogenase (LDH) levels at timepoints after the administration of atest oligomeric compound are compared to baseline values obtained priorto administration, to those of control animals that did not receive testoligomeric compound, or to values known to be associated with normalanimals from previous experiments (historical controls) or fromliterature.

b. Modulation of the Amount or Activity of Off-Target Genes In Vivo

In certain embodiments the modulation of the amount or activity ofoff-target genes in vivo may be determined by microarray analysis. Afteradministration of an oligomeric compound to an animal in vivo or a groupof animals in vivo, one or more of the animals may be sacrificed and thetissue analyzed by microarray to determine expression levels of a largenumber of specific genes, or even the entire genome (genome profiling).In certain embodiments, one or more of the animals may be sacrificed andthe tissue analyzed by microarray analysis at various times (e.g., 0, 24hours, 48 hours, 72 hours, 96 hours) after administration of anoligomeric compound. Such microarray analysis may be compared to similaranalyses from untreated animals and/or from animals treated with adifferent oligomeric compound.

In certain embodiments, toxixity of treated animals is assessed atvarious times. In certain embodiments, the tissue of the sacrificedanimals is analyzed for indications of toxicity by any method known tothose having skill in the art. In certain embodiments, any animals notsacrificed for microarray analysis may continue to be observed forindications of acute toxicity at various time points, for example at 24hours, 48 hours, 72 hours, and 96 hours after administration.

In certain embodiments, the degree of the change in expression ofcertain off-target genes as determined by microarray analysis may becorrelated with some measure of toxicity. In certain embodiments, thedegree of the decrease in expression of certain off-target genes may becorrelated with increase in AST levels or ALT levels. In certainembodiments, after microarray analysis, the degree of the increase inexpression of certain off-target genes may be correlated with the amountof increase in some measure of toxicity, for example, AST levels or ALTlevels. After correlation between the in vivo modulation of the amountor activity of an off-target gene and in vivo toxicity is performed, theoff-target genes may be sorted by the coefficient of determination fromhighest to lowest. In this manner off-target genes may be identifiedwhere the in vivo modulation of the amount or activity of a genecorrelates strongly with some measure of toxicity (sentinel genes), forexample AST levels or ALT levels. In certain embodiments, off-targetgenes having the strongest correlation between a decrease in in vivoexpression and toxicity may be identified as sentinel genes. In certainembodiments, off-target genes having the strongest correlation between adecrease in in vivo expression and increase in AST levels may beidentified as sentinel genes. In certain embodiments, off-target geneshaving the strongest correlation between a decrease in in vivoexpression and increase in ALT levels may be identified sentinel genes.In certain embodiments, off-target genes having the strongestcorrelation between an increase in expression in vivo and toxicity maybe identified sentinel genes. In certain embodiments, off-target geneshaving the strongest correlation between an increase in in vivoexpression and increase in ALT levels may be identified as sentinelgenes. In certain embodiments, off-target genes having the strongestcorrelation between an increase in in vivo expression and increase inAST levels may be identified as sentinel genes.

In certain embodiments, the modulation of the amount or activity ofoff-target genes may be correlated with one or more measure of toxicity.One having skill in the art may correlate the modulation of the amountor activity of off-target genes with one or more measure of toxicityusing any statistical method known to those having skill in the art. Incertain embodiments, the correlation of the modulation of the amount oractivity of off-target genes with one or more measure of toxicity isassessed by calculating the coefficient of determination. In certainembodiments, the correlation of the modulation of the amount or activityof off-target genes may be correlated with one or more measure oftoxicity by using the coefficient of determination, r². In this mannersentinel genes may be identified where the in vivo modulation of theamount or activity of an off-target gene in response to an oligomericcompound correlates strongly with some measure of toxicity.

In certain embodiments the degree of the decrease in expression of anoff-target gene may be correlated with an increase in AST levels. Incertain embodiments the degree of the decrease in expression of anoff-target gene may be correlated with an increase in ALT levels. Incertain embodiments the degree of the decrease in expression of anoff-target gene may be correlated with an increase in ALP levels. Incertain embodiments the degree of the decrease in expression of anoff-target gene may be correlated with an increase in TBIL levels. Incertain embodiments the degree of the decrease in expression of anoff-target gene may be correlated with an increase in albumin levels. Incertain embodiments the degree of the decrease in expression of anoff-target gene may be correlated with an increase in serum glucoselevels. In certain embodiments the degree of the decrease in expressionof an off-target gene may be correlated with an increase in LDH levels.

In certain embodiments the degree of the increase in expression of anoff-target gene may be correlated with an increase in AST levels. Incertain embodiments the degree of the increase in expression of anoff-target gene may be correlated with an increase in ALT levels. Incertain embodiments the degree of the increase in expression of anoff-target gene may be correlated with an increase in ALP levels. Incertain embodiments the degree of the increase in expression of anoff-target gene may be correlated with an increase in TBIL levels. Incertain embodiments the degree of the increase in expression of anoff-target gene may be correlated with an increase in albumin levels. Incertain embodiments the degree of the increase in expression of anoff-target gene may be correlated with an increase in serum glucoselevels. In certain embodiments the degree of the increase in expressionof an off-target gene may be correlated with an increase in LDH levels.

In certain embodiments the degree of the decrease in expression of asentinel gene may be correlated with an increase in AST levels. Incertain embodiments the degree of the decrease in expression of asentinel gene may be correlated with an increase in ALT levels. Incertain embodiments the degree of the decrease in expression of asentinel gene may be correlated with an increase in ALP levels. Incertain embodiments the degree of the decrease in expression of asentinel gene may be correlated with an increase in TBIL levels. Incertain embodiments the degree of the decrease in expression of asentinel gene may be correlated with an increase in albumin levels. Incertain embodiments the degree of the decrease in expression of asentinel gene may be correlated with an increase in serum glucoselevels. In certain embodiments the degree of the decrease in expressionof a sentinel gene may be correlated with an increase in LDH levels.

In certain embodiments the degree of the increase in expression of asentinel gene may be correlated with an increase in AST levels. Incertain embodiments the degree of the increase in expression of asentinel gene may be correlated with an increase in ALT levels. Incertain embodiments the degree of the increase in expression of asentinel gene may be correlated with an increase in ALP levels. Incertain embodiments the degree of the increase in expression of asentinel gene may be correlated with an increase in TBIL levels. Incertain embodiments the degree of the increase in expression of asentinel gene may be correlated with an increase in albumin levels. Incertain embodiments the degree of the increase in expression of asentinel gene may be correlated with an increase in serum glucoselevels. In certain embodiments the degree of the increase in expressionof a sentinel gene may be correlated with an increase in LDH levels.

In certain embodiments, any number of off-target genes may be rankedaccording to coefficient of determination, r², between toxicity and thedegree modulation of the amount or activity of off-target geneexpression. In certain embodiments, any number of off-target genes maybe ranked according to the strength of correlation between toxicity asmeasured by ALT levels and the degree of the decrease in off-targetexpression. In certain embodiments, any number of off-target genes maybe ranked according to the strength of correlation between toxicity asmeasured by AST levels and the degree the decrease in off-target geneexpression.

In certain embodiments, any number of sentinel genes may be rankedaccording to coefficient of determination, r², between toxicity and thedegree modulation of the amount or activity of sentinel gene expression.In certain embodiments, any number of sentinel genes may be rankedaccording to the strength of correlation between toxicity as measured byALT levels and the degree of the decrease in sentinel expression. Incertain embodiments, any number of sentinel genes may be rankedaccording to the strength of correlation between toxicity as measured byAST levels and the degree the decrease in sentinel gene expression.

In certain embodiments, the 1 to 150 or more off-target genes having thestrongest in vivo correlation between a decrease in expression andtoxicity may be identified as sentinel genes. In certain embodiments,the 1 to 100 off-target genes having the strongest in vivo correlationbetween a decrease in expression and toxicity may be identified assentinel genes. In certain embodiments, the 1 to 50 off-target geneshaving the strongest in vivo correlation between a decrease inexpression and toxicity may be identified as sentinel genes. In certainembodiments, the 1 to 40 off-target genes having the strongestcorrelation between a decrease in expression and toxicity may beidentified as sentinel genes. In certain embodiments, the 1 to 30off-target genes having the strongest correlation between a decrease inexpression and toxicity may be identified as sentinel genes. In certainembodiments, the 1 to 20 off-target genes having the strongestcorrelation between a decrease in expression and toxicity may beidentified as sentinel genes. In certain embodiments, the 1 to 10off-target genes having the strongest correlation between a decrease inexpression and toxicity may be identified as sentinel genes. In certainembodiments, the 1 to 5 off-target genes having the strongestcorrelation between a decrease in expression and toxicity may beidentified as sentinel genes.

In certain embodiments, the 1 to 150 or more off-target genes having thestrongest correlation between an increase in expression and toxicity maybe identified as sentinel genes. In certain embodiments, the 1 to 100off-target genes having the strongest correlation between an increase inexpression and toxicity may be identified as sentinel genes. In certainembodiments, the 1 to 50 off-target genes having the strongestcorrelation between an increase in expression and toxicity may beidentified as sentinel genes. In certain embodiments, the 1 to 40off-target genes having the strongest correlation between an increase inexpression and toxicity may be identified as sentinel genes. In certainembodiments, the 1 to 30 off-target genes having the strongestcorrelation between an increase in expression and toxicity may beidentified as sentinel genes. In certain embodiments, the 1 to 20off-target genes having the strongest correlation between an increase inexpression and toxicity may be identified as sentinel genes. In certainembodiments, the 1 to 10 off-target genes having the strongestcorrelation between an increase in expression and toxicity may beidentified as sentinel genes. In certain embodiments, the 1 to 5off-target genes having the strongest correlation between an increase inexpression and toxicity may be identified as sentinel genes.

The methods described herein enable one having skill in the art to thenidentify any number of off-target genes, sentinel genes, or transcripts.The methods described herein enable one having skill in the art to thenidentify any number of off-target genes, sentinel genes, or transcriptswherein the decrease in expression of the sentinel gene or transcript iscorrelated to some measure of toxicity. The methods described hereinenable one having skill in the art to then identify any number ofoff-target genes, sentinel genes, or transcripts wherein the increase inexpression of the sentinel gene or transcript is correlated to somemeasure of toxicity. In this manner, one having skill in the art mayidentify any number of off-target genes, sentinel genes, or transcriptsaccording to the correlation between the modulation of the amount oractivity of the off-target genes, sentinel genes, or transcripts in vivoand any measure of toxicity. In certain embodiments, the off-targetgenes, sentinel genes, or transcripts identified may be used for furtherin vitro evaluation to develop a sub-set of in vitro off-target genes,sentinel genes, or transcripts that correlate to in vivo toxicity. Incertain embodiments, at least one antisense compound that is predictednot to be toxic in vivo is made and then tested in an animal.

In certain embodiments, sentinel genes are identified empirically. Forexample, in certain embodiments, oligomeric compounds that modulate theamount or activity of a particular off target gene in vitro are found tocause toxicity when administered in vivo. Such observed correlationbetween modulation of the amount or activity of an off-target gene invitro and corresponding in vivo toxicity does not necessarily indicatethat modulation of the amount or activity of the off target gene is thecause of the observed toxicity. Indeed, an off-target gene might noteven be modulated in vivo. The utility of the observation, however, isindependent of mechanism. Regardless of causation, oligomeric compoundsthat modulate the amount or activity of a strongly correlated off-targetsentinel gene are predicted to be toxic in vivo. In certain embodiments,homology between an oligomeric compound and a sentinel gene does notresult in the modulation of the amount or activity of said sentinelgene. In certain embodiments, homology between an oligomeric compoundand an off-target gene does not result in the modulation of the amountor activity of said off target gene. In certain embodiments, anoligomeric compound modulates the amount or activity of an off-targetgene without hybridizing to said off-target gene. In certainembodiments, an oligomeric compound modulates the amount or activity ofa sentinel gene without hybridizing to said sentinel gene.

c. Modulation of the Amount or Activity of Off-Target Genes In Vivo

In certain embodiments, the modulation of the amount or activity of anynumber of off-target genes in response to any given oligonucleotide maybe measured in vitro. Any suitable cell lines that express genes ofinterest may be used for the in vitro screen. In certain embodimentshepatocyte cell lines may be used. In certain embodiments, BEND celllines may be used. In certain embodiments, HeLa cell lines may be used.In certain embodiments HepG2 cell lines may be used. The degree ofmodulation of the amount or activity of the off-target genes in-vitromay then be compared with off-target genes that have been identified asbeing modulated in vivo. In this manner, off-target genes that have ahigh amount of modulation of amount or activity in vivo and which alsohave a high amount of modulation of amount or activity in vitro may beidentified. In certain embodiments, the modulation of the amount oractivity of off-target genes identified as having a high correlationbetween measurements of acute toxicity and a decrease in expression invivo may be correlated with the degree of a decrease in expression invitro. In certain embodiments, the modulation of the amount or activityof off-target genes identified as having a high correlation betweenacute toxicity and an increase in expression in vivo may be correlatedwith the modulation of amount or activity in vitro. In certainembodiments, certain off-target genes may be identified that have a highcorrelation between a change in the modulation of amount or activity invivo and a change in the modulation of amount or activity in vitro. Forexample, in certain embodiments, certain off-target genes identified asdemonstrating a relatively strong decrease in expression in vivo, willalso demonstrate a relatively strong decrease in expression in vitro. Incertain embodiments, the identification of such in vitro off-targetgenes, for example, genes that demonstrate a decrease in expression upontransfection with a given oligomeric compound, will then predict adecrease in expression of the same off-target genes in vivo, andtherefore will predict toxicity in vivo. Once in vitro off-target genesare identified, then any oligomeric compound maybe screened in vitro bytransfecting a cell with the oligomeric compound and measuring themodulation of the amount or activity of one or more identifiedoff-target genes. In some embodiments, this method reduces the need foran acute single-dose in vivo screen for most oligomeric compounds.

Any method known to those having skill in the art may be used to measurethe modulation of the amount or activity of the off-target genes invitro. In certain embodiments, cells may be transfected with oligomericcompounds using electroporation. Other suitable transfection reagentsknown in the art include, but are not limited to, CYTOFECTIN™,LIPOFECTAMINE™, OLIGOFECTAMINE™, and FUGENE™ In certain embodiments,RT-PCR is used to measure the modulation of the amount or activity ofoff-target genes in vitro.

d. Certain Off-Target Genes

In certain embodiments, the modulation of the amount or activity of oneor more off-target genes in vitro is used to determine the toxicity ofan oligomeric compound in vivo. In certain embodiments the decrease inexpression of one or more off-target genes in vitro is used to determinethe toxicity of an oligomeric compound in vivo. In certain embodimentsthe increase in expression of one or more off-target genes in vitro isused to determine the toxicity of an oligomeric compound in vivo.

In certain embodiments, the amount of the decrease in expression invitro of one or more of the off-target genes listed in Table 1 below maybe used to determine the toxicity of an oligomeric compound in vivo.

TABLE 1 In Vitro Off-Target Genes Symbol Official Name Adcy9 adenylatecyclase 9 Ptprk protein tyrosine phosphatase, receptor type, K Tbc1d22aTBC1 domain family, member 22a Exoc6b exocyst complex component 6B Ftofat mass and obesity associated RAPTOR regulatory associated protein ofMTOR, complex 1 Iqgap2 IQ motif containing GTPase activating protein 2Vti1a vesicle transport through interaction with t-SNAREs homolog 1ABC057079 cDNA sequence BC057079 Fbx117 F-box and leucine-rich repeatprotein 17 Bre brain and reproductive organ-expressed protein Cgnl1cingulin-like 1 Msi2 Musashi homolog 2 (Drosophila) Mcph1 microcephaly,primary autosomal recessive 1 Atxn1 ataxin 1 Vps13b vacuolar proteinsorting 13B (yeast) Cadps2 Ca2+-dependent activator protein forsecretion 2 Ppp3ca protein phosphatase 3, catalytic subunit, alphaisoform Ppm11 protein phosphatase 1 (formerly 2C)-like Ubac2 ubiquitinassociated domain containing 2 Bcas3 breast carcinoma amplified sequence3 Gphn gephyrin Atp9b ATPase, class II, type 9B Chn2 chimerin(chimaerin) 2 Fars2 phenylalanine-tRNA synthetase 2 (mitochondrial) Adkadenosine kinase Odz3 odd Oz/ten-m homolog 3 (Drosophila) Macrod1 MACROdomain containing 1 Atg10 Autophagy-related protein 10 Fggy carbohydratekinase domain containing Vps53 vacuolar protein sorting 53 homolog (S.cerevisiae) Itpr2 inositol 1,4,5-triphosphate receptor, type 20610012H03Rik Riken cDNA 0610012H03 gene

In certain embodiments, the degree of the increase in expression invitro of one or more of the off-target genes listed in Table 2 below maybe used to determine the toxicity of an oligomeric compound in vivo.

TABLE 2 In Vitro Off-Target Genes Symbol Gene ID Rassf1 56289 Dus4171916 Mdm2 17246 Brp16 59053 0610010K14Rik 104457 Rce1 19671 Ilf2 67781Setd1a 233904 Gar1 68147 FAM203A 59053

In certain embodiments, the amount of the decrease in expression invitro of one or more of the off-target genes listed in Table 3 below maybe used to determine the toxicity of an oligomeric compound in vivo.

TABLE 3 In Vitro Off-Target Genes Symbol Gene ID Rsrc1 66880 Cadps2320405 Aprin 100710 Faf1 14084 Sntg2 268534 Odz3 23965 St3gal3 20441Sox5 20678 BC033915 70661 A530050D06Rik 104816 Fbxl17 50758 Msi2 76626Pard3 93742 4933407C03Rik 74440 Itpr1 16438 Zdhhc14 224454 Rrbp1 81910Mtmr14 97287 Dpyd 99586 Ptprd 19266 Pcca 110821 Lmf1 76483 Iqgap2 544963Centg2 347722 Btbd9 224671 Ubac2 68889 Ptprk 19272 R3hdm2 71750 Psme4103554 Ppp3ca 19055 Vps53 68299 Vps13b 666173 Mgl1 23945 Chn2 69993Atxn1 20238 Acot7 70025 Lpp 210126 Itpr2 16439 Mapkap1 227743 Stx8 55943Ghr 14600 Bcas3 192197 Exoc6b///Sec1512 75914 9030420J04Rik 71544 Pck118534 Ube2e2 218793 Pik3c2g 18705 1300010F03Rik 219189 Apbb2 11787 Mcph1244329 Sergef 27414 Adcy9 11515 Pkp4 227937 Ascc3 77987 Enpp2 18606Sel11 20338 Macrod1 107227 Vti1a 53611 Wdr7 104082 4932417H02Rik 74370Bach2 12014 0610012H03Rik 74088 Adk 11534 Dym 69190 Pitpnm2 19679Slc41a2 338365 Fgfr2 14183 Bre 107976 Gphn 268566 Mical3 194401 Fars269955 Ap3b1 11774 Vps13a 271564 Skap2 54353 Sds 231691 Coval///Enox2209224 Pitpnc1 71795 Large 16795 Lrba 80877 Atg10 66795 Atp9b 50771 Cask12361 Ppm11 242083 Alcam 11658 Atg7 74244 Nfia 18027 Supt3h 109115 Med2768975 Cgnl1 68178 Dennd1a 227801 Smoc1 64075 Prkca 18750 2210408F21Rik73652 Map2k5 23938 Dock4 238130 LOC100036521 100036521 Sil1 81500Tbc1d22a 223754 2310009E04Rik 75578 BC057079 230393 Fhit 14198 Uvrag78610 Dtnb 13528 Fto 26383 Immp21 93757

In certain embodiments, the measurement of the modulation of the amountor activity of more than one off-target gene in vitro may increase theprobability of predicting toxicity in vivo. For example, in certainembodiments, a cell is transfected with an oligomeric compound ofinterest and then the modulation of the amount or activity of two ormore off-target genes is measured. In such embodiments, if bothoff-target genes are modulated then there is a higher probability thatthe oligomeric compound of interest is toxic than if only one of the twooff-target genes were modulated. In certain embodiments, a cell istransfected with an oligomeric compound of interest and then themodulation of the amount or activity of three or more off-target genesis measured. In such embodiments, if all three off-target genes aremodulated then there is a higher probability that the oligomericcompound of interest is toxic than if only one of the three off-targetgenes were modulated. In certain embodiments, a cell is transfected withan oligomeric compound of interest and then the modulation of the amountor activity of four or more off-target genes is measured. In suchembodiments, if all four off-target genes are modulated or if two of thefour off-target genes are modulated or if three of the four off-targetgenes are modulated then there is a higher probability that theoligomeric compound of interest is toxic than if only one of the fouroff-target genes were modulated. Likewise, in certain embodiments, ifthree of the four off-target genes were modulated then there is a higherprobability that the oligomeric compound of interest is toxic than ifonly one of the four off-target genes were modulated or if two of thefour off-target genes were modulated. In certain embodiments, a cell istransfected with an oligomeric compound of interest and then themodulation of the amount or activity of five or more off-target genes ismeasured. In such embodiments, if all five off-target genes aremodulated or if two of the five off-target genes are modulated or ifthree of the five or four of the five off-target genes are modulatedthen there is a higher probability that the oligomeric compound ofinterest is toxic than if only one of the four off-target genes weremodulated. In certain embodiments, a cell is transfected with anoligomeric compound of interest and then the modulation of the amount oractivity of at least six off-target genes is measured. In suchembodiments, if all six off-target genes are modulated or if two of thesix off-target genes are modulated or if three of the six or four of thesix or five of the six off-target genes are modulated then there is ahigher probability that the oligomeric compound of interest is toxicthan if only one of the four off-target genes were modulated.

In certain embodiments the off-target gene is Adcy9. In certainembodiments the off-target gene is Ptprk. In certain embodiments theoff-target gene is Tbc1d22a. In certain embodiments the off-target geneis Exoc6b. In certain embodiments the off-target gene is Fto. In certainembodiments the off-target gene is RAPTOR. In certain embodiments theoff-target gene is Iqgap2. In certain embodiments the off-target gene isVti1a. In certain embodiments the off-target gene is BC057079. Incertain embodiments the off-target gene is Fbx117. In certainembodiments the off-target gene is Bre. In certain embodiments theoff-target gene is Cgn11. In certain embodiments the off-target gene isMsi2. In certain embodiments the off-target gene is Mcph1. In certainembodiments the off-target gene is Atxn1. In certain embodiments theoff-target gene is Vps13b. In certain embodiments the off-target gene isCadps2. In certain embodiments the off-target gene is Ppp3ca. In certainembodiments the off-target gene is Ppm11. In certain embodiments theoff-target gene is Ubac2. In certain embodiments the off-target gene isBcas3. In certain embodiments the off-target gene is Gphn. In certainembodiments the off-target gene is Atp9b. In certain embodiments theoff-target gene is Chn2 In certain embodiments the off-target gene isFars2. In certain embodiments the off-target gene is Adk. In certainembodiments the off-target gene is Odz3. In certain embodiments theoff-target gene is Macrod1. In certain embodiments the off-target geneis Atg10. In certain embodiments the off-target gene is Fggy. In certainembodiments the off-target gene is Vps53. In certain embodiments theoff-target gene is Itpr2. In certain embodiments the off-target gene is0610012H03Rik.

In certain embodiments the off-target gene is Rassf1. In certainembodiments the off-target gene is Dus41. In certain embodiments theoff-target gene is Mdm2. In certain embodiments the off-target gene isBrp16. In certain embodiments the off-target gene is 0610010K14Rik. Incertain embodiments the off-target gene is Rce1. In certain embodimentsthe off-target gene is I1f2. In certain embodiments the off-target geneis Setd1a. In certain embodiments the off-target gene is Gar1. Incertain embodiments the off-target gene is FAM203A.

In certain embodiments the off-target gene is Rsrc1. In certainembodiments the off-target gene is Cadps2. In certain embodiments theoff-target gene is Aprin. In certain embodiments the off-target gene isFaf1. In certain embodiments the off-target gene is Sntg2. In certainembodiments the off-target gene is Odz3. In certain embodiments theoff-target gene is St3ga13. In certain embodiments the off-target geneis Sox5. In certain embodiments the off-target gene is BC033915. Incertain embodiments the off-target gene is A530050D06Rik. In certainembodiments the off-target gene is Fbx117. In certain embodiments theoff-target gene is Msi2. In certain embodiments the off-target gene isPard3. In certain embodiments the off-target gene is 4933407C03Rik. Incertain embodiments the off-target gene is Itpr1. In certain embodimentsthe off-target gene is Zdhhc14. In certain embodiments the off-targetgene is Rrbp1. In certain embodiments the off-target gene is Mtmr14. Incertain embodiments the off-target gene is Dpyd. In certain embodimentsthe off-target gene is Ptprd. In certain embodiments the off-target geneis Pcca. In certain embodiments the off-target gene is Lmf1. In certainembodiments the off-target gene is Iqgap2. In certain embodiments theoff-target gene is Centg2. In certain embodiments the off-target gene isBtbd9. In certain embodiments the off-target gene is Ubac2. In certainembodiments the off-target gene is Ptprk. In certain embodiments theoff-target gene is R3hdm2. In certain embodiments the off-target gene isPsme4. In certain embodiments the off-target gene is Ppp3ca. In certainembodiments the off-target gene is Vps53. In certain embodiments theoff-target gene is Vps13b. In certain embodiments the off-target gene isMg11. In certain embodiments the off-target gene is Chn2 In certainembodiments the off-target gene is Atxn1. In certain embodiments theoff-target gene is Acot7. In certain embodiments the off-target gene isLpp. In certain embodiments the off-target gene is Itpr2. In certainembodiments the off-target gene is Mapkap1. In certain embodiments theoff-target gene is Stx8. In certain embodiments the off-target gene isGhr. In certain embodiments the off-target gene is Bcas3. In certainembodiments the off-target gene is Exoc6b///Sec1512. In certainembodiments the off-target gene is 9030420J04Rik.

In certain embodiments the off-target gene is Pck1. In certainembodiments the off-target gene is Ube2e2. In certain embodiments theoff-target gene is Pik3c2g. In certain embodiments the off-target geneis 1300010F03Rik. In certain embodiments the off-target gene is Apbb2.In certain embodiments the off-target gene is Mcph1. In certainembodiments the off-target gene is Sergef. In certain embodiments theoff-target gene is Adcy9. In certain embodiments the off-target gene isPkp4. In certain embodiments the off-target gene is Ascc3. In certainembodiments the off-target gene is Enpp2. In certain embodiments theoff-target gene is Se111. In certain embodiments the off-target gene isMacrod1. In certain embodiments the off-target gene is Vti1a. In certainembodiments the off-target gene is Wdr7. In certain embodiments theoff-target gene is 4932417H02Rik. In certain embodiments the off-targetgene is Bach2. In certain embodiments the off-target gene is0610012H03Rik. In certain embodiments the off-target gene is Adk. Incertain embodiments the off-target gene is Dym. In certain embodimentsthe off-target gene is Pitpnm2. In certain embodiments the off-targetgene is S1c41a2. In certain embodiments the off-target gene is Fgfr2. Incertain embodiments the off-target gene is Bre. In certain embodimentsthe off-target gene is Gphn. In certain embodiments the off-target geneis Mica13. In certain embodiments the off-target gene is Fars2. Incertain embodiments the off-target gene is Ap3b1. In certain embodimentsthe off-target gene is Vps13a. In certain embodiments the off-targetgene is Skap2. In certain embodiments the off-target gene is Sds. Incertain embodiments the off-target gene is Cova1///Enox2. In certainembodiments the off-target gene is Pitpnc1. In certain embodiments theoff-target gene is Large. In certain embodiments the off-target gene isLrba.

In certain embodiments the off-target gene is Atg10. In certainembodiments the off-target gene is Atp9b. In certain embodiments theoff-target gene is Cask. In certain embodiments the off-target gene isPpm11. In certain embodiments the off-target gene is A1cam. In certainembodiments the off-target gene is Atg7. In certain embodiments theoff-target gene is Nfia. In certain embodiments the off-target gene isSupt3h. In certain embodiments the off-target gene is Med27. In certainembodiments the off-target gene is Cgn11. In certain embodiments theoff-target gene is Dennd1a. In certain embodiments the off-target geneis Smoc1. In certain embodiments the off-target gene is Prkca. Incertain embodiments the off-target gene is 2210408F21Rik. In certainembodiments the off-target gene is Map2k5. In certain embodiments theoff-target gene is Dock4. In certain embodiments the off-target gene isLOC100036521. In certain embodiments the off-target gene is Si11. Incertain embodiments the off-target gene is Tbc1d22a. In certainembodiments the off-target gene is 2310009E04Rik. In certain embodimentsthe off-target gene is BC057079. In certain embodiments the off-targetgene is Fhit. In certain embodiments the off-target gene is Uvrag. Incertain embodiments the off-target gene is Dtnb. In certain embodimentsthe off-target gene is Fto. In certain embodiments the off-target geneis Immp21.

In certain embodiments the off-target gene is 4932417H02Rik. In certainembodiments the off-target gene is mKIAA0919///Sec1512///Exoc6b. Incertain embodiments the off-target gene is Fbx117. In certainembodiments the off-target gene is Chn2 In certain embodiments theoff-target gene is Fto. In certain embodiments the off-target gene isAK053274///mKIAA0532//Vps13b///AK049111. In certain embodiments theoff-target gene is Lrba///Lba. In certain embodiments the off-targetgene is Fars2. In certain embodiments the off-target gene is Pomt2. Incertain embodiments the off-target gene is Wwc1. In certain embodimentsthe off-target gene is Atg10. In certain embodiments the off-target geneis Gng12. In certain embodiments the off-target gene is Smg6. In certainembodiments the off-target gene is 2310008H04Rik. In certain embodimentsthe off-target gene is Ptprk. In certain embodiments the off-target geneis Cadps2. In certain embodiments the off-target gene is Supt3h. Incertain embodiments the off-target gene is St3ga13. In certainembodiments the off-target gene is Atg7. In certain embodiments theoff-target gene is Fggy. In certain embodiments the off-target gene isUbe2e2. In certain embodiments the off-target gene is Immp21. In certainembodiments the off-target gene is Bcas3. In certain embodiments theoff-target gene is Mnat1. In certain embodiments the off-target gene isItpr2. In certain embodiments the off-target gene is Adcy9. In certainembodiments the off-target gene is S1c17a2. In certain embodiments theoff-target gene is Sergef. In certain embodiments the off-target gene isSmoc1. In certain embodiments the off-target gene is Dym. In certainembodiments the off-target gene is Nfia. In certain embodiments theoff-target gene is Odz3. In certain embodiments the off-target gene isEnox2. In certain embodiments the off-target gene is Tbc1d5. In certainembodiments the off-target gene is BC057079. In certain embodiments theoff-target gene is Cob1. In certain embodiments the off-target gene isMsi2. In certain embodiments the off-target gene is Esr1. In certainembodiments the off-target gene is Dexi. In certain embodiments theoff-target gene is AA536749. In certain embodiments the off-target geneis Efna5. In certain embodiments the off-target gene is Med27. Incertain embodiments the off-target gene is Cdka11. In certainembodiments the off-target gene is Atp9b. In certain embodiments theoff-target gene is Igfbp4. In certain embodiments the off-target gene isSaa4. In certain embodiments the off-target gene is Fry1. In certainembodiments the off-target gene is Mica13///Kiaa0819.

In certain embodiments the off-target gene is Itpr1. In certainembodiments the off-target gene is AK031097///Ppm11. In certainembodiments the off-target gene is Pard3. In certain embodiments theoff-target gene is Mgmt. In certain embodiments the off-target gene isMtmr14. In certain embodiments the off-target gene is Pik3c2g. Incertain embodiments the off-target gene is Fndc3b. In certainembodiments the off-target gene is Cask. In certain embodiments theoff-target gene is Galnt10. In certain embodiments the off-target geneis Tbc1d22a. In certain embodiments the off-target gene is Macrod1. Incertain embodiments the off-target gene is Clec16a. In certainembodiments the off-target gene is Dis312. In certain embodiments theoff-target gene is Cyp2j9. In certain embodiments the off-target gene isSntg2. In certain embodiments the off-target gene is Si11. In certainembodiments the off-target gene is 1300010F03Rik. In certain embodimentsthe off-target gene is Cux1. In certain embodiments the off-target geneis 1110012L19Rik. In certain embodiments the off-target gene is Prnpip1.In certain embodiments the off-target gene is Atxn1. In certainembodiments the off-target gene is Gpr39. In certain embodiments theoff-target gene is Ghr. In certain embodiments the off-target gene isPtprd. In certain embodiments the off-target gene is Errfi1. In certainembodiments the off-target gene is AK137808///Gtde1. In certainembodiments the off-target gene is Atp11c. In certain embodiments theoff-target gene is Prkag2. In certain embodiments the off-target gene isLrit1. In certain embodiments the off-target gene is Tnrc6b. In certainembodiments the off-target gene is Cgn11. In certain embodiments theoff-target gene is Large. In certain embodiments the off-target gene isGphn. In certain embodiments the off-target gene is Bbs9. In certainembodiments the off-target gene is Pcx. In certain embodiments theoff-target gene is mKIAA1188///Clmn. In certain embodiments theoff-target gene is Pet1121. In certain embodiments the off-target geneis Stxbp5. In certain embodiments the off-target gene is Ext2. Incertain embodiments the off-target gene is Dtnbp 1. In certainembodiments the off-target gene is Arsb. In certain embodiments theoff-target gene is Zdhhc14. In certain embodiments the off-target geneis Mbn12. In certain embodiments the off-target gene is Dtnb. In certainembodiments the off-target gene is Pitpnm2.

In certain embodiments the off-target gene is Herc2. In certainembodiments the off-target gene is Enpp2. In certain embodiments theoff-target gene is Vti1a. In certain embodiments the off-target gene isDock4///mKIAA0716. In certain embodiments the off-target gene is Dpyd.In certain embodiments the off-target gene is Arsg. In certainembodiments the off-target gene is Pcca. In certain embodiments theoff-target gene is Snd1. In certain embodiments the off-target gene isCcdc91. In certain embodiments the off-target gene is Acsm5. In certainembodiments the off-target gene is Gtf2i. In certain embodiments theoff-target gene is S1c39a11. In certain embodiments the off-target geneis Adarb1. In certain embodiments the off-target gene is Pcnx. Incertain embodiments the off-target gene is Zcchc7. In certainembodiments the off-target gene is Bbs4. In certain embodiments theoff-target gene is Uroc1. In certain embodiments the off-target gene isCdh2. In certain embodiments the off-target gene is Map2k2. In certainembodiments the off-target gene is BC038349. In certain embodiments theoff-target gene is 5033414K04Rik. In certain embodiments the off-targetgene is Epb4.1. In certain embodiments the off-target gene is Dock1. Incertain embodiments the off-target gene is Pank1. In certain embodimentsthe off-target gene is S1c4a4. In certain embodiments the off-targetgene is Tmtc2. In certain embodiments the off-target gene is Ncrna00153.In certain embodiments the off-target gene is BC099512. In certainembodiments the off-target gene is Farp1. In certain embodiments theoff-target gene is Nfib. In certain embodiments the off-target gene isArhgef11. In certain embodiments the off-target gene is Got1. In certainembodiments the off-target gene is Cables1. In certain embodiments theoff-target gene is Elov15. In certain embodiments the off-target gene isUsp20. In certain embodiments the off-target gene is Myo9b. In certainembodiments the off-target gene is Nedd41///mKIAA0439. In certainembodiments the off-target gene is 0610012H03Rik. In certain embodimentsthe off-target gene is D430042009Rik. In certain embodiments theoff-target gene is Ehbp1. In certain embodiments the off-target gene isTtc7b. In certain embodiments the off-target gene is Se111. In certainembodiments the off-target gene is Vps13a///CHAC.

In certain embodiments the off-target gene is Ddb2. In certainembodiments the off-target gene is Rnf213. In certain embodiments theoff-target gene is Myo1e. In certain embodiments the off-target gene isMasp2. In certain embodiments the off-target gene is Gfra1. In certainembodiments the off-target gene is Hsd17b2. In certain embodiments theoff-target gene is Rapgef6///mKIAA4052. In certain embodiments theoff-target gene is Ascc3///AK144867. In certain embodiments theoff-target gene is Prkca. In certain embodiments the off-target gene isParva. In certain embodiments the off-target gene is Fert2. In certainembodiments the off-target gene is Stau2. In certain embodiments theoff-target gene is Mapkap1. In certain embodiments the off-target geneis AK140547///Ralgps1. In certain embodiments the off-target gene isSox5. In certain embodiments the off-target gene is Chdh. In certainembodiments the off-target gene is Smad3. In certain embodiments theoff-target gene is Skap2. In certain embodiments the off-target gene isMad1///Mad111. In certain embodiments the off-target gene is Pdzrn3. Incertain embodiments the off-target gene is Arid1b. In certainembodiments the off-target gene is Aspg. In certain embodiments theoff-target gene is Anxa6. In certain embodiments the off-target gene isArfgef1. In certain embodiments the off-target gene is Hs6st1. Incertain embodiments the off-target gene is Arhgap26///mKIAA0621. Incertain embodiments the off-target gene is Wdr7.

In certain embodiments the off-target gene is B230342M21Rik///N4bp211.In certain embodiments the off-target gene is Asph. In certainembodiments the off-target gene is Iqgap2. In certain embodiments theoff-target gene is Ugcg11. In certain embodiments the off-target gene isBC033915. In certain embodiments the off-target gene ismKIAA0665///Rab11fip3. In certain embodiments the off-target gene isSox6. In certain embodiments the off-target gene is Fbxo31. In certainembodiments the off-target gene is Ubac2. In certain embodiments theoff-target gene is Hmgn3. In certain embodiments the off-target gene is4930402H24Rik. In certain embodiments the off-target gene is Foxp1. Incertain embodiments the off-target gene is Cd9912. In certainembodiments the off-target gene is C530044N13Rik///Cpped1. In certainembodiments the off-target gene is Trappc9///1810044A24Rik. In certainembodiments the off-target gene is Rabgap11. In certain embodiments theoff-target gene is Tb11x. In certain embodiments the off-target gene isHs2st1. In certain embodiments the off-target gene is Tmem16k///Ano10.In certain embodiments the off-target gene is Agap1. In certainembodiments the off-target gene is Map2k5. In certain embodiments theoff-target gene is Susd4. In certain embodiments the off-target gene isRbms1///AK011205. In certain embodiments the off-target gene is Gig18.In certain embodiments the off-target gene is 4933407C03Rik///mKIAA1694.In certain embodiments the off-target gene is Oaf. In certainembodiments the off-target gene is Cadm1. In certain embodiments theoff-target gene is Tsc2. In certain embodiments the off-target gene isZbtb20. In certain embodiments the off-target gene is Aig1. In certainembodiments the off-target gene is Zfp277///AK172713.

In certain embodiments the off-target gene is Nsmaf. In certainembodiments the off-target gene is Ppp1ca. In certain embodiments theoff-target gene is Vav2. In certain embodiments the off-target gene isMg11. In certain embodiments the off-target gene is Ppnr. In certainembodiments the off-target gene is 2310007H09Rik. In certain embodimentsthe off-target gene is M113. In certain embodiments the off-target geneis Peli2. In certain embodiments the off-target gene is Spag9///JSAP2.In certain embodiments the off-target gene is Ctnna1. In certainembodiments the off-target gene is Ostf1. In certain embodiments theoff-target gene is 11-Sep. In certain embodiments the off-target gene isMan2a1. In certain embodiments the off-target gene is N1k. In certainembodiments the off-target gene is AU040829. In certain embodiments theoff-target gene is Apbb2. In certain embodiments the off-target gene isNsmce2. In certain embodiments the off-target gene is Btbd9. In certainembodiments the off-target gene is Rap1gds1. In certain embodiments theoff-target gene is Cry11. In certain embodiments the off-target gene isS1co2a1. In certain embodiments the off-target gene is Ubr1. In certainembodiments the off-target gene is Lrrc16a///Lac16. In certainembodiments the off-target gene is Mon2. In certain embodiments theoff-target gene is Fbxw7. In certain embodiments the off-target gene isPpp3ca.

In certain embodiments the off-target gene is AK040794///Acaca. Incertain embodiments the off-target gene is Man1a. In certain embodimentsthe off-target gene is Rbms3. In certain embodiments the off-target geneis Adipor2. In certain embodiments the off-target gene is Ryr3. Incertain embodiments the off-target gene is Tpk1. In certain embodimentsthe off-target gene is Pepd. In certain embodiments the off-target geneis C2cd21. In certain embodiments the off-target gene is Akap7. Incertain embodiments the off-target gene is BC030307. In certainembodiments the off-target gene is Fam149b. In certain embodiments theoff-target gene is Spop. In certain embodiments the off-target gene isXrcc4. In certain embodiments the off-target gene is Dip2c. In certainembodiments the off-target gene is 1700009P17Rik. In certain embodimentsthe off-target gene is Pdia5. In certain embodiments the off-target geneis Pck1. In certain embodiments the off-target gene is Vps53. In certainembodiments the off-target gene is Eefsec. In certain embodiments theoff-target gene is Pb1d. In certain embodiments the off-target gene isDennd1a. In certain embodiments the off-target gene is Ncoa1. In certainembodiments the off-target gene is Fign.

In certain embodiments the off-target gene is 4933421E11Rik. In certainembodiments the off-target gene is Rpusd4. In certain embodiments theoff-target gene is AK019895///Chchd8. In certain embodiments theoff-target gene is Ange12. In certain embodiments the off-target gene isThumpd3. In certain embodiments the off-target gene is Polr2d. Incertain embodiments the off-target gene is Gadd45a. In certainembodiments the off-target gene is Ece2. In certain embodiments theoff-target gene is 2310009B15Rik. In certain embodiments the off-targetgene is 1110002N22Rik. In certain embodiments the off-target gene isSetd1a. In certain embodiments the off-target gene is 2810432D09Rik. Incertain embodiments the off-target gene is Serbp1. In certainembodiments the off-target gene is 2310039H08Rik. In certain embodimentsthe off-target gene is Mtap1s. In certain embodiments the off-targetgene is Plek2. In certain embodiments the off-target gene is Bola1. Incertain embodiments the off-target gene is AK172713///9430016H08Rik. Incertain embodiments the off-target gene is 1700052N19Rik. In certainembodiments the off-target gene is Rnf6. In certain embodiments theoff-target gene is Thtpa. In certain embodiments the off-target gene isOrmd11. In certain embodiments the off-target gene is 2900026A02Rik. Incertain embodiments the off-target gene is Polr2a. In certainembodiments the off-target gene is Ywhah. In certain embodiments theoff-target gene is Krt18. In certain embodiments the off-target gene isZfp518b. In certain embodiments the off-target gene is Spryd4. Incertain embodiments the off-target gene is 0610010K14Rik. In certainembodiments the off-target gene is AU021838///Mipo11. In certainembodiments the off-target gene is Adam32. In certain embodiments theoff-target gene is 2810422020Rik. In certain embodiments the off-targetgene is Lgals3bp. In certain embodiments the off-target gene is Ltv1. Incertain embodiments the off-target gene is Fand1. In certain embodimentsthe off-target gene is 0610007P22Rik. In certain embodiments theoff-target gene is Sf3b4. In certain embodiments the off-target gene isFermt2. In certain embodiments the off-target gene is Znhit3. In certainembodiments the off-target gene is Znf746. In certain embodiments theoff-target gene is Trnau1ap. In certain embodiments the off-target geneis Rp113. In certain embodiments the off-target gene is Rp124. Incertain embodiments the off-target gene is Pdgfa. In certain embodimentsthe off-target gene is Tmem41a. In certain embodiments the off-targetgene is Cep78. In certain embodiments the off-target gene is I1f2. Incertain embodiments the off-target gene is 2510049J12Rik. In certainembodiments the off-target gene is Ap4b1. In certain embodiments theoff-target gene is Ppp1r11. In certain embodiments the off-target geneis Arfgap2. In certain embodiments the off-target gene is Aldoc.

In certain embodiments the off-target gene is Hus1. In certainembodiments the off-target gene is Ppp2r1a. In certain embodiments theoff-target gene is Setd6. In certain embodiments the off-target gene isAK036897///Trex1. In certain embodiments the off-target gene is Rpp38.In certain embodiments the off-target gene is Nars. In certainembodiments the off-target gene is Mrp150. In certain embodiments theoff-target gene is Mthfd2. In certain embodiments the off-target gene is2010321M09Rik. In certain embodiments the off-target gene is Lrrc57. Incertain embodiments the off-target gene is Cox18. In certain embodimentsthe off-target gene is Umps. In certain embodiments the off-target geneis Prdx3. In certain embodiments the off-target gene is Usp18. Incertain embodiments the off-target gene is Isgf3g. In certainembodiments the off-target gene is No111. In certain embodiments theoff-target gene is Brf2. In certain embodiments the off-target gene isPpid. In certain embodiments the off-target gene is Myadm. In certainembodiments the off-target gene is Krt8. In certain embodiments theoff-target gene is Avpi1. In certain embodiments the off-target gene isRab3d. In certain embodiments the off-target gene is Hn1. In certainembodiments the off-target gene is Ino80b. In certain embodiments theoff-target gene is 2310016C08Rik. In certain embodiments the off-targetgene is Gtf3a. In certain embodiments the off-target gene is Srrt. Incertain embodiments the off-target gene is Nsbp1. In certain embodimentsthe off-target gene is Polr2h. In certain embodiments the off-targetgene is Tommy. In certain embodiments the off-target gene is S1c1a4. Incertain embodiments the off-target gene is Bxdc2. In certain embodimentsthe off-target gene is Gemin4. In certain embodiments the off-targetgene is Gb1. In certain embodiments the off-target gene isC87414///AA792892. In certain embodiments the off-target gene isAK052711. In certain embodiments the off-target gene is Ddx52. Incertain embodiments the off-target gene is Commd3. In certainembodiments the off-target gene is Shmt2. In certain embodiments theoff-target gene is Tmem97. In certain embodiments the off-target gene isSp5. In certain embodiments the off-target gene is Gar1. In certainembodiments the off-target gene is Esco2. In certain embodiments theoff-target gene is 2310047B19Rik. In certain embodiments the off-targetgene is Pop7.

In certain embodiments the off-target gene is Plrg1. In certainembodiments the off-target gene is Cct4. In certain embodiments theoff-target gene is Cc19. In certain embodiments the off-target gene isPnp1. In certain embodiments the off-target gene is Etaa1. In certainembodiments the off-target gene is Prss8. In certain embodiments theoff-target gene is Rce1. In certain embodiments the off-target gene isUsp22. In certain embodiments the off-target gene is Ruvb12. In certainembodiments the off-target gene is Impdh2. In certain embodiments theoff-target gene is Npb. In certain embodiments the off-target gene isExosc2. In certain embodiments the off-target gene is Dus41. In certainembodiments the off-target gene is 1700029J07Rik. In certain embodimentsthe off-target gene is 1700123020Rik. In certain embodiments theoff-target gene is Nudt2. In certain embodiments the off-target gene isGltpd1. In certain embodiments the off-target gene is Dbr1. In certainembodiments the off-target gene is Ins16. In certain embodiments theoff-target gene is Rps4x. In certain embodiments the off-target gene isCcdc51. In certain embodiments the off-target gene is Mrto4. In certainembodiments the off-target gene is Gde1. In certain embodiments theoff-target gene is Hexim2. In certain embodiments the off-target gene isAtmin. In certain embodiments the off-target gene is Ms11. In certainembodiments the off-target gene is Qars. In certain embodiments theoff-target gene is Dak. In certain embodiments the off-target gene isCcrk. In certain embodiments the off-target gene is Armc6. In certainembodiments the off-target gene is 2810008M24Rik. In certain embodimentsthe off-target gene is Kdelc1///1700029F09Rik. In certain embodimentsthe off-target gene is Srd5a3. In certain embodiments the off-targetgene is Hirip3. In certain embodiments the off-target gene isA430005L14Rik. In certain embodiments the off-target gene is BC026590.In certain embodiments the off-target gene is Cldn3///Wbscr25. Incertain embodiments the off-target gene is Zfp637. In certainembodiments the off-target gene is Fen1. In certain embodiments theoff-target gene is Alg5. In certain embodiments the off-target gene isAls2cr2///Stradb.

In certain embodiments the off-target gene is Rp129. In certainembodiments the off-target gene is Tmub1. In certain embodiments theoff-target gene is Rp18. In certain embodiments the off-target gene isZfp161. In certain embodiments the off-target gene is D4Wsu114e. Incertain embodiments the off-target gene is Ddx28. In certain embodimentsthe off-target gene is Npm1. In certain embodiments the off-target geneis Nkrf. In certain embodiments the off-target gene is 1110058L19Rik. Incertain embodiments the off-target gene is Snapc4. In certainembodiments the off-target gene is Nme3. In certain embodiments theoff-target gene is Peo1. In certain embodiments the off-target gene isRp119. In certain embodiments the off-target gene is Pbx2. In certainembodiments the off-target gene is 2210411K11Rik. In certain embodimentsthe off-target gene is Rps10. In certain embodiments the off-target geneis Rps8. In certain embodiments the off-target gene is No16. In certainembodiments the off-target gene is Rps21. In certain embodiments theoff-target gene is Hsd3b4. In certain embodiments the off-target gene isParp16. In certain embodiments the off-target gene is Palm. In certainembodiments the off-target gene is Trip6. In certain embodiments theoff-target gene is Acot6. In certain embodiments the off-target gene isAbhd14a. In certain embodiments the off-target gene is Mrp140. Incertain embodiments the off-target gene is Rps12. In certain embodimentsthe off-target gene is Ptrh2. In certain embodiments the off-target geneis Trim21. In certain embodiments the off-target gene is Necap1. Incertain embodiments the off-target gene is Ythdc1. In certainembodiments the off-target gene is Gpn3. In certain embodiments theoff-target gene is Sfrs6. In certain embodiments the off-target gene isENSMUSG00000059775///Rps26. In certain embodiments the off-target geneis Nup43. In certain embodiments the off-target gene is Rnps1. Incertain embodiments the off-target gene is Psip1. In certain embodimentsthe off-target gene is Btbd6. In certain embodiments the off-target geneis Cdkn2aipn1. In certain embodiments the off-target gene is Rp17. Incertain embodiments the off-target gene is Eif2b4. In certainembodiments the off-target gene is Psma4. In certain embodiments theoff-target gene is Zscan12. In certain embodiments the off-target geneis Rp131. In certain embodiments the off-target gene is Kbtbd7. Incertain embodiments the off-target gene is Dtwd1. In certain embodimentsthe off-target gene is 4930473A06Rik///AK029637. In certain embodimentsthe off-target gene is Mfap3. In certain embodiments the off-target geneis Ccdc130. In certain embodiments the off-target gene is Cdc34. Incertain embodiments the off-target gene is Ifi30. In certain embodimentsthe off-target gene is Chac2. In certain embodiments the off-target geneis Ufsp1.

In certain embodiments the off-target gene is Gemin6. In certainembodiments the off-target gene is Igtp. In certain embodiments theoff-target gene is Ankrd49. In certain embodiments the off-target geneis AK206957///AK050697. In certain embodiments the off-target gene isCcdc32. In certain embodiments the off-target gene isENSMUSG00000053178. In certain embodiments the off-target gene is Rccd1.In certain embodiments the off-target gene is Med11. In certainembodiments the off-target gene is 2810416G20Rik. In certain embodimentsthe off-target gene is F8a. In certain embodiments the off-target geneis Adat2. In certain embodiments the off-target gene is Sat1. In certainembodiments the off-target gene is Zcchc8. In certain embodiments theoff-target gene is Pnrc2. In certain embodiments the off-target gene isTmem129. In certain embodiments the off-target gene is Mrps22. Incertain embodiments the off-target gene is 4930572J05Rik. In certainembodiments the off-target gene is Rp112. In certain embodiments theoff-target gene is Ino80c. In certain embodiments the off-target gene isCdca7. In certain embodiments the off-target gene is Usp11. In certainembodiments the off-target gene is BC031781. In certain embodiments theoff-target gene is 2200002D01Rik. In certain embodiments the off-targetgene is Hexim1. In certain embodiments the off-target gene is Thns11.

In certain embodiments the off-target gene is AK009724. In certainembodiments the off-target gene is Thyn1///mThy28. In certainembodiments the off-target gene is Prpf6. In certain embodiments theoff-target gene is Med21. In certain embodiments the off-target gene isWbp5. In certain embodiments the off-target gene is Iars. In certainembodiments the off-target gene is Mfsd10. In certain embodiments theoff-target gene is Nt5dc2. In certain embodiments the off-target gene is2010003K11Rik. In certain embodiments the off-target gene is Rpp21. Incertain embodiments the off-target gene is Gimap1. In certainembodiments the off-target gene is Rassf7. In certain embodiments theoff-target gene is Scrn2. In certain embodiments the off-target gene isCd3eap. In certain embodiments the off-target gene is Ccdc85b. Incertain embodiments the off-target gene is AK087382. In certainembodiments the off-target gene is Psmg1. In certain embodiments theoff-target gene is Atic. In certain embodiments the off-target gene isTmem179b. In certain embodiments the off-target gene is Kbtbd4. Incertain embodiments the off-target gene is Tmem60. In certainembodiments the off-target gene is 2810026P18Rik. In certain embodimentsthe off-target gene is Zfp213. In certain embodiments the off-targetgene is Psmg2. In certain embodiments the off-target gene is AA881470.In certain embodiments the off-target gene is Eef1d. In certainembodiments the off-target gene is Chchd5. In certain embodiments theoff-target gene is Ube216. In certain embodiments the off-target gene isGstm4. In certain embodiments the off-target gene is Taf1a. In certainembodiments the off-target gene is S1c26a1. In certain embodiments theoff-target gene is Era11. In certain embodiments the off-target gene isMrp115///AK017820. In certain embodiments the off-target gene is Ccdc23.In certain embodiments the off-target gene is Fb1. In certainembodiments the off-target gene is C130022K22Rik. In certain embodimentsthe off-target gene is L00554292. In certain embodiments the off-targetgene is Mrps18b. In certain embodiments the off-target gene is Tmem177.In certain embodiments the off-target gene is Brp16. In certainembodiments the off-target gene is Tlcd2. In certain embodiments theoff-target gene is Rdh14. In certain embodiments the off-target gene isTmem185b. In certain embodiments the off-target gene is Rp135. Incertain embodiments the off-target gene is Mrp111. In certainembodiments the off-target gene is Ythdf2. In certain embodiments theoff-target gene is Pdcd2. In certain embodiments the off-target gene isEif2s3x. In certain embodiments the off-target gene is Aldoa.

In certain embodiments the off-target gene is Kat2a. In certainembodiments the off-target gene is Rdm1. In certain embodiments theoff-target gene is Rp1p2. In certain embodiments the off-target gene is2610301G19Rik. In certain embodiments the off-target gene is Rp13. Incertain embodiments the off-target gene is Tnnc1. In certain embodimentsthe off-target gene is Pgam1. In certain embodiments the off-target geneis Smug1. In certain embodiments the off-target gene is 2310004I24Rik.In certain embodiments the off-target gene is Sap30. In certainembodiments the off-target gene is 1500012F01Rik. In certain embodimentsthe off-target gene is Sf3b3. In certain embodiments the off-target geneis Tagap///Tagap 1. In certain embodiments the off-target gene is Ripk4.In certain embodiments the off-target gene is BC160215///Ids. In certainembodiments the off-target gene is Cbr4. In certain embodiments theoff-target gene is Usp42. In certain embodiments the off-target gene isTrp53. In certain embodiments the off-target gene is Psmb6. In certainembodiments the off-target gene is Tapbp1. In certain embodiments theoff-target gene is Jtv1. In certain embodiments the off-target gene isKhsrp. In certain embodiments the off-target gene is Oas11. In certainembodiments the off-target gene is Hgs. In certain embodiments theoff-target gene is Rps20. In certain embodiments the off-target gene isH2afx. In certain embodiments the off-target gene is Psmb4. In certainembodiments the off-target gene is Tgm1. In certain embodiments theoff-target gene is Daxx. In certain embodiments the off-target gene isClk2///Scamp3. In certain embodiments the off-target gene is Sfrs7. Incertain embodiments the off-target gene is S1c35a4. In certainembodiments the off-target gene is Chtf8. In certain embodiments theoff-target gene is Fiz1. In certain embodiments the off-target gene isSnrnp25. In certain embodiments the off-target gene is Taxlbp1. Incertain embodiments the off-target gene is Rcan3. In certain embodimentsthe off-target gene is Scnm1. In certain embodiments the off-target geneis Coi1. In certain embodiments the off-target gene is Cog8. In certainembodiments the off-target gene is Cdk4. In certain embodiments theoff-target gene is Lsm2. In certain embodiments the off-target gene isKlf6. In certain embodiments the off-target gene is Cct8. In certainembodiments the off-target gene is Tmem107. In certain embodiments theoff-target gene is Noc21. In certain embodiments the off-target gene isArmc10. In certain embodiments the off-target gene is C430004E15Rik. Incertain embodiments the off-target gene is Rangrf. In certainembodiments the off-target gene is Kbtbd2. In certain embodiments theoff-target gene is Impact. In certain embodiments the off-target gene isRnmt11. In certain embodiments the off-target gene is Fnta. In certainembodiments the off-target gene is Srxn1. In certain embodiments theoff-target gene is Rpp14. In certain embodiments the off-target gene isAK003073. In certain embodiments the off-target gene is Rp115.

In certain embodiments the off-target gene is ENSMUSG00000074747. Incertain embodiments the off-target gene is Casp2. In certain embodimentsthe off-target gene is 6330503K22Rik. In certain embodiments theoff-target gene is Xaf1. In certain embodiments the off-target gene isPus1. In certain embodiments the off-target gene is Rnf187. In certainembodiments the off-target gene is 2610024G14Rik. In certain embodimentsthe off-target gene is Mrps23. In certain embodiments the off-targetgene is Mat2a. In certain embodiments the off-target gene is Eif5. Incertain embodiments the off-target gene is Fem1b. In certain embodimentsthe off-target gene is Rp118. In certain embodiments the off-target geneis Mrps30. In certain embodiments the off-target gene is Rp128. Incertain embodiments the off-target gene is Otub1. In certain embodimentsthe off-target gene is Mapk6. In certain embodiments the off-target geneis T1r6. In certain embodiments the off-target gene is Rps24. In certainembodiments the off-target gene is Eif4a1. In certain embodiments theoff-target gene is Pigp. In certain embodiments the off-target gene isRars. In certain embodiments the off-target gene is Pyroxd1. In certainembodiments the off-target gene is Pabpc4. In certain embodiments theoff-target gene is Rps19. In certain embodiments the off-target gene isMrps16. In certain embodiments the off-target gene is Abcf2. In certainembodiments the off-target gene is Rilp12. In certain embodiments theoff-target gene is Thoc1. In certain embodiments the off-target gene isGpatch4. In certain embodiments the off-target gene is AK009175. Incertain embodiments the off-target gene is Eif2b2.

C. METHODS OF PREDICTING IN VITRO OR IN VIVO TOXICITY

In certain embodiments, a computer or any other means may be used todetermine the amount of sequence complementarity between the nucleobasesequence of any oligomeric compound and the nucleobase sequence of anyoff-target gene. In certain embodiments, a computer or any other meansmay be used to determine the amount of sequence complementarity betweenthe nucleobase sequence of any oligomeric compound and the nucleobasesequence of any sentinel gene. In certain embodiments, oligomericcompounds having high amounts of complementarity between theirnucleobase sequence and any number of off-target genes and/or sentinelgenes may indicate toxicity. In certain embodiments, one having skill inthe art may select a minimum amount of complementarity between thenucleobase sequence of the oligomeric compound and the nucleobasesequence of any given off-target gene and/or sentinel gene. In certainembodiments, the nucleobase sequence of an oligomeric compound may have90% complementarity with the nucleobase sequence of an off-target geneand/or sentinel gene. In certain embodiments, the nucleobase sequence ofan oligomeric compound may have 100% complementarity with the nucleobasesequence of an off-target gene and/or sentinel gene.

In certain embodiments, the nucleobase sequence of an oligomericcompound may have 1 to 2 mismatches relative to the nucleobase sequenceof an off-target gene and/or sentinel gene. In certain embodiments, thenucleobase sequence of an oligomeric compound may have 1 mismatchrelative to the nucleobase sequence of an off-target gene and/orsentinel gene. In certain embodiments, the nucleobase sequence of anoligomeric compound may have 2 mismatches relative to the nucleobasesequence of an off-target gene and/or sentinel gene.

In certain embodiments, after one having skill in the art has selected aminimum amount of complementarity between the nucleobase sequence of theoligomeric compound and the nucleobase sequence of any given off-targetgene and/or sentinel gene, the number of off-target genes and/orsentinel genes in a genome having an equal to or greater amount ofcomplementarity with the oligomeric compound may be identified. Incertain embodiments, before one having skill in the art has selected aminimum amount of complementarity between the nucleobase sequence of theoligomeric compound and the nucleobase sequence of any given off-targetgene and/or sentinel gene, the total number of off-target genes and/orsentinel genes in a genome having an equal to or greater amount ofcomplementarity with the oligomeric compound may be identified. In someembodiments, a computer is used to identify the number of off-targetgenes and/or sentinel gene in a genome that have an equal to or greateramount of complementarity with the oligomeric compound.

In certain embodiments, the total number of off-target genes and/orsentinel genes having an equal to or greater amount of complementaritywith the oligomeric compound may be identified. In certain embodiments,the greater the number of off-target genes and/or sentinel genes havingan equal to or greater amount of complementarity with the oligomericcompound indicates greater probability of in vitro and in vivo toxicity.

D. OLIGOMERIC COMPOUNDS

Certain methods disclosed herein provide for the identification ofoligomeric compounds. In certain embodiments, the methods disclosedherein may be used to discover novel non-toxic oligomeric compounds. Incertain embodiments, the methods disclosed herein may be used todiscover novel non-toxic oligomer modifications or oligomer motifs. Incertain embodiments, at least one oligomeric compounds that is predictednot to be toxic in vivo is made and then tested in an animal.

In certain embodiments, the present invention provides oligomericcompounds. In certain embodiments, such oligomeric compounds compriseoligonucleotides optionally comprising one or more conjugate and/orterminal groups. In certain embodiments, an oligomeric compound consistsof an oligonucleotide. In certain embodiments, oligonucleotides compriseone or more chemical modifications. Such chemical modifications includemodifications of one or more nucleoside (including modifications to thesugar moiety and/or the nucleobase) and/or modifications to one or moreinternucleoside linkage.

a. Certain Modified Nucleosides

In certain embodiments, provided herein are oligomeric compoundscomprising or consisting of oligonuleotides comprising at least onemodified nucleoside. Such modified nucleosides comprise a modified sugarmoeity, a modified nucleobase, or both a modified sugar moiety and amodified nucleobase.

i. Certain Modified Sugar Moieties

In certain embodiments, compounds of the invention comprise one or moremodified nucleosides comprising a modified sugar moiety. Such compoundscomprising one or more sugar-modified nucleosides may have desirableproperties, such as enhanced nuclease stability or increased bindingaffinity with a target nucleic acid relative to an oligonucleotidecomprising only nucleosides comprising naturally occurring sugarmoieties. In certain embodiments, modified sugar moieties aresubstituted sugar moieties. In certain embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, 0-C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certainembodiments, substituted sugars comprise more than one non-bridgingsugar substituent, for example, 2′-F-5′-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—;4′-CH₂-2′,4′-(CH₂)₂-2′,4′-(CH₂)₃-2′,4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, andanalogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15,2008); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g.,WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogsthereof (see, e.g., WO2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004);4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)-0-2′-, wherein each R is,independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,(J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA, and (K) Ethylene(methoxy)(4′-(CH(CH₂OMe)-O-2′) BNA (also referred to as constrained MOE or cMOE)as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfer atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran. Such tetrahydropyrans may befurther modified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ areeach, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆alkynyl; and

each of R₁ and R₂ is independently selected from among: hydrogen,halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′,2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, the present invention provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In certainembodiments, oligonucleotides comprise one or more RNA-like nucleosides.In certain embodiments, oligonucleotides comprise one or more DNA-likenucleotides.

b. Certain Modified Nucleobases

In certain embodiments, nucleosides of the present invention compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present invention comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazinecytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

c. Certain Internucleoside Linkages

In certain embodiments, nucleosides may be linked together using anyinternucleoside linkage to form oligonucleotides. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂A thiodiester(—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)₂—O—);and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Modified linkages,compared to natural phosphodiester linkages, can be used to alter,typically increase, nuclease resistance of the oligonucleotide. Incertain embodiments, internucleoside linkages having a chiral atom canbe prepared as a racemic mixture, or as separate enantiomers.Representative chiral linkages include, but are not limited to,alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

d. Certain Motifs

In certain embodiments, oligomeric compounds comprise or consist ofoligonucleotides. In certain embodiments, such oligonucleotides compriseone or more chemical modification. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified sugars. Incertain embodiments, chemically modified oligonucleotides comprise oneor more modified nucleobases. In certain embodiments, chemicallymodified oligonucleotides comprise one or more modified internucleosidelinkages. In certain embodiments, the chemical modifications (sugarmodifications, nucleobase modifications, and/or linkage modifications)define a pattern or motif. In certain embodiments, the patterns ofchemical modifications of sugar moieties, internucleoside linkages, andnucleobases are each independent of one another. Thus, anoligonucleotide may be described by its sugar modification motif,internucleoside linkage motif and/or nucleobase modification motif (asused herein, nucleobase modification motif describes the chemicalmodifications to the nucleobases independent of the sequence ofnucleobases).

e. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar motif. Such sugar motifs include but are not limited to any ofthe sugar modifications discussed herein.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar motif, which comprises two external regionsor “wings” and a central or internal region or “gap.” The three regionsof a gapmer sugar motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar motifs of the two wings are the same as oneanother (symmetric sugar gapmer). In certain embodiments, the sugarmotifs of the 5′-wing differs from the sugar motif of the 3′-wing(asymmetric sugar gapmer).

i. Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certainembodiments, each nucleobase is modified. In certain embodiments, noneof the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, oligonucleotides comprise one or morenucleosides comprising a modified nucleobase. In certain embodiments,oligonucleotides having a gapmer sugar motif comprise a nucleosidecomprising a modified nucleobase. In certain such embodiments, onenucleoside comprising a modified nucleobases is in the central gap of anoligonucleotide having a gapmer sugar motif. In certain embodiments, thesugar is an unmodified 2′ deoxynucleoside. In certain embodiments, themodified nucleobase is selected from: a 2-thio pyrimidine and a5-propyne pyrimidine

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

ii. Certain Nucleoside Motifs

In certain embodiments, oligonucleotides comprise nucleosides comprisingmodified sugar moieties and/or nucleosides comprising modifiednucleobases. Such motifs can be described by their sugar motif and theirnucleobase motif separately or by their nucleoside motif, which providespositions or patterns of modified nucleosides (whether modified sugar,nucleobase, or both sugar and nucleobase) in an oligonucleotide.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer nucleoside motif, which comprises two externalregions or “wings” and a central or internal region or “gap.” The threeregions of a gapmer nucleoside motif (the 5′-wing, the gap, and the3′-wing) form a contiguous sequence of nucleosides wherein at least someof the sugar moieties and/or nucleobases of the nucleosides of each ofthe wings differ from at least some of the sugar moieties and/ornucleobase of the nucleosides of the gap. Specifically, at least thenucleosides of each wing that are closest to the gap (the 3′-mostnucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing)differ from the neighboring gap nucleosides, thus defining the boundarybetween the wings and the gap. In certain embodiments, the nucleosideswithin the gap are the same as one another. In certain embodiments, thegap includes one or more nucleoside that differs from one or more othernucleosides of the gap. In certain embodiments, the nucleoside motifs ofthe two wings are the same as one another (symmetric gapmer). In certainembodiments, the nucleoside motifs of the 5′-wing differs from thenucleoside motif of the 3′-wing (asymmetric gapmer).

1. Certain 5′-Wings

In certain embodiments, the 5′-wing of a gapmer consists of 1 to 5linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 2 to 5 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 4 or 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of1 to 4 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 to 3 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 1 or 2 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 2 to 4 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of2 or 3 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 3 or 4 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 1 nucleoside. In certainembodiments, the 5′-wing of a gapmer consists of 2 linked nucleosides.In certain embodiments, the 5′-wing of a gapmer consists of 3 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 5 linked nucleosides.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmercomprises at least two bicyclic nucleosides. In certain embodiments, the5′-wing of a gapmer comprises at least three bicyclic nucleosides. Incertain embodiments, the 5′-wing of a gapmer comprises at least fourbicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 5′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 5′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 5′-wing ofa gapmer comprises at least one 2′-substituted nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one 2′-OMe nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a non-bicyclic modified nucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is a2′-substituted nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments,each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 5′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer has a nucleoside motifselected from among the following: ADDA; ABDAA; ABBA; ABB; ABAA; AABAA;AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB;AAAAA; ABBDC; ABDDC; ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB;AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB,wherein each A is a modified nucleoside of a first type, each B is amodified nucleoside of a second type, each C is a modified nucleoside ofa third type, and each D is an unmodified deoxynucleoside.

In certain embodiments, an oligonucleotide comprises any 5′-wing motifprovided herein. In certain such embodiments, the oligonucleotide is a5′-hemimer (does not comprise a 3′-wing). In certain embodiments, suchan oligonucleotide is a gapmer. In certain such embodiments, the 3′-wingof the gapmer may comprise any nucleoside motif.

2. Certain 3′-Wings

In certain embodiments, the 3′-wing of a gapmer consists of 1 to 5linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 2 to 5 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 4 or 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of1 to 4 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 to 3 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 1 or 2 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 2 to 4 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of2 or 3 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 3 or 4 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 1 nucleoside. In certainembodiments, the 3′-wing of a gapmer consists of 2 linked nucleosides.In certain embodiments, the 3′-wing of a gapmer consists of 3linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 5 linked nucleosides.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 3′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 3′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least two non-bicyclic modified nucleosides. Incertain embodiments, the 3′-wing of a gapmer comprises at least threenon-bicyclic modified nucleosides. In certain embodiments, the 3′-wingof a gapmer comprises at least four non-bicyclic modified nucleosides.In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-substituted nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is anon-bicyclic modified nucleoside. In certain embodiments, eachnucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is a2′-MOE nucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneLNA nucleoside and at least one non-bicyclic modified nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-substituted nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside and atleast one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one LNA nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one non-bicyclic modified nucleoside, andat least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least one constrained ethyl nucleoside, at leastone non-bicyclic modified nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one LNA nucleoside, at least one non-bicyclicmodified nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-substituted nucleoside, and atleast one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one constrained ethyl nucleoside, at least one2′-substituted nucleoside, and at least one 2′-deoxynucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside, at least one 2′-substituted nucleoside, and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-MOEnucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-OMenucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer has a nucleoside motifselected from among the following: ABB; ABAA; AAABAA, AAAAABAA; AABAA;AAAABAA; AAABAA; ABAB; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB;AAAA; AAA; AA; AB; ABBB; ABAB; AABBB; wherein each A is a modifiednucleoside of a first type, each B is a modified nucleoside of a secondtype. In certain embodiments, an oligonucleotide comprises any 3′-wingmotif provided herein. In certain such embodiments, the oligonucleotideis a 3′-hemimer (does not comprise a 5′-wing). In certain embodiments,such an oligonucleotide is a gapmer. In certain such embodiments, the5′-wing of the gapmer may comprise any nucleoside motif.

3. Certain Central Regions (Gaps)

In certain embodiments, the gap of a gapmer consists of 6 to 20 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6to 15 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 to 12 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 6 to 10 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 6 to 8 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6or 7 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 7 to 10 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 7 to 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 8 to 10 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 8or 9 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 linked nucleosides. In certain embodiments, the gap of agapmer consists of 7 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 8 linked nucleosides. In certain embodiments,the gap of a gapmer consists of 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 10 linked nucleosides. Incertain embodiments, the gap of a gapmer consists of 11 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 12linked nucleosides.

In certain embodiments, each nucleoside of the gap of a gapmer is a2′-deoxynucleoside. In certain embodiments, the gap comprises one ormore modified nucleosides. In certain embodiments, each nucleoside ofthe gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleosidethat is “DNA-like.” In such embodiments, “DNA-like” means that thenucleoside has similar characteristics to DNA, such that a duplexcomprising the gapmer and an RNA molecule is capable of activating RNaseH. For example, under certain conditions, 2′-(ara)-F have been shown tosupport RNase H activation, and thus is DNA-like. In certainembodiments, one or more nucleosides of the gap of a gapmer is not a2′-deoxynucleoside and is not DNA-like. In certain such embodiments, thegapmer nonetheless supports RNase H activation (e.g., by virtue of thenumber or placement of the non-DNA nucleosides).

In certain embodiments, gaps comprise a stretch of unmodified2′-deoxynucleoside interrupted by one or more modified nucleosides, thusresulting in three sub-regions (two stretches of one or more2′-deoxynucleosides and a stretch of one or more interrupting modifiednucleosides). In certain embodiments, no stretch of unmodified2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certainembodiments, such short stretches is achieved by using short gapregions. In certain embodiments, short stretches are achieved byinterrupting a longer gap region.

4. Certain Gapmer Motifs

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′wing, wherein the 5′-wing, gap, and 3′ wing are independently selectedfrom among those discussed above. For example, in certain embodiments, agapmer has a 5′-wing, a gap, and a 3′-wing having features selected fromamong those listed in the following non-limiting table:

TABLE 4 Certain Gapmer Nucleoside Motifs 5′-wing region Central gapregion 3′-wing region ADDA DDDDDD ABB ABBA DDDADDDD ABAA AAAAAAADDDDDDDDDDD AAA AAAAABB DDDDDDDD BBAAAAA ABB DDDDADDDD ABB ABB DDDDBDDDDBBA ABB DDDDDDDDD BBA AABAA DDDDDDDDD AABAA ABB DDDDDD AABAA AAABAADDDDDDDDD AAABAA AAABAA DDDDDDDDD AAB ABAB DDDDDDDDD ABAB AAABB DDDDDDDBBA ABADB DDDDDDD BBA ABA DBDDDDDDD BBA ABA DADDDDDDD BBA ABAB DDDDDDDDBBA AA DDDDDDDD BBBBBBBB ABB DDDDDD ABADB AAAAB DDDDDDD BAAAA ABBBDDDDDDDDD AB AB DDDDDDDDD BBBA ABBB DDDDDDDDD BBBA AB DDDDDDDD ABA ABBDDDDWDDDD BBA AAABB DDDWDDD BBAAA ABB DDDDWWDDD BBA ABADB DDDDDDD BBAABBDC DDDDDDD BBA ABBDDC DDDDDD BBA ABBDCC DDDDDD BBA ABB DWWDWWDWW BBAABB DWDDDDDDD BBA ABB DDWDDDDDD BBA ABB DWWDDDDDD BBA AAABB DDWDDDDDD AABB DDWDWDDDD BBABBBB ABB DDDD(^(N)D)DDDD BBA AAABB DDD(^(N)D)DDD BBAAAABB DDDD(^(N)D)(^(N)D)DDD BBA ABBD(^(N)D)(^(N)D)D(^(N)D)(^(N)D)D(^(N)D)(^(N)D) BBA ABB D(^(N)D)DDDDDDDBBA ABB DD(^(N)D)DDDDDD BBA ABB D(^(N)D)(^(N)D)DDDDDD BBA AAABBDD(^(N)D)DDDDDD AA BB DD(^(N)D)D(^(N)D)DDDD BBABBBBwherein each A is a modified nucleoside of a first type, each B is amodified nucleoside of a second type and each W is a modified nucleosideof either the first type, the second type or a third type, each D is anucleoside comprising an unmodified 2′ deoxy sugar moiety and unmodifiednucleobase, and ^(N)D is modified nucleoside comprising a modifiednucleobase and an unmodified 2′ deoxy sugar moiety.

In certain embodiments, each A comprises a modified sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moietyselected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each A comprises a bicyclic sugar moiety. In certainembodiments, each A comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each A comprises a modified nucleobase. In certainembodiments, each A comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.

In certain embodiments, each B comprises a modified sugar moiety. Incertain embodiments, each B comprises a 2′-substituted sugar moiety. Incertain embodiments, each B comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each B comprises a bicyclic sugar moiety. In certainembodiments, each B comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each B comprises a modified nucleobase. In certainembodiments, each B comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne urindine nucleoside.

In certain embodiments, each C comprises a modified sugar moiety. Incertain embodiments, each C comprises a 2′-substituted sugar moiety. Incertain embodiments, each C comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each C comprises a 5′-substituted sugar moiety. In certainembodiments, each C comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Ccomprises a bicyclic sugar moiety. In certain embodiments, each Ccomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises amodified nucleobase. In certain embodiments, each C comprises a modifiednucleobase selected from among 2-thio-thymidine and 5-propyne uridine.

In certain embodiments, each W comprises a modified sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each W comprises a 5′-substituted sugar moiety. In certainembodiments, each W comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Wcomprises a bicyclic sugar moiety. In certain embodiments, each Wcomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each W comprises asugar surrogate. In certain embodiments, each W comprises a sugarsurrogate selected from among HNA and F-HNA.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, and the other comprises a 2′-substituted sugar moiety. Incertain embodiments, one of A or B is an LNA nucleoside and the other ofA or B comprises a 2′-substituted sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside and the other of A or B comprises a2′-substituted sugar moiety. In certain embodiments, one of A or B is anα-L-LNA nucleoside and the other of A or B comprises a 2′-substitutedsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-MOE sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-MOE sugar moiety. In certain embodiments, one of A or Bis an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOEsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-F sugar moiety. In certain embodiments, one of A or B isan α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugarmoiety. In certain embodiments, one of A or B is an LNA nucleoside andthe other of A or B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A orB is an α-L-LNA nucleoside and the other of A or B comprises a2′-(ara)-F sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-substituted sugar moiety. In certain embodiments, A is anLNA nucleoside and B comprises a 2′-substituted sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-substitutedsugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-substituted sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-MOE sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-MOE sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-F sugar moiety. In certain embodiments,A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certainembodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugarmoiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-MOE sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-F sugar moiety. In certain embodiments,B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certainembodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugarmoiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-(ara)-F sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, another of A or B comprises a 2′-substituted sugar moietyand W comprises a modified nucleobase. In certain embodiments, one of Aor B is an LNA nucleoside, another of A or B comprises a 2′-substitutedsugar moiety, and W comprises a modified nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-substituted sugar moiety, and C comprises a modifiednucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-substituted sugar moiety,and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a modified nucleobase. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 2-thio-thymidine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and C comprises a 5-propyne uridine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, andC comprises a 5-propyne uridine nucleobase. In certain embodiments, oneof A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridinenucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is anα-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an LNA nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a sugar surrogate. In certain embodiments, one of A or Bis an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a sugar HNA surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aHNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a F-HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-MeDNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety.

In certain embodiments, at least two of A, B or W comprises a2′-substituted sugar moiety, and the other comprises a bicyclic sugarmoiety. In certain embodiments, at least two of A, B or W comprises abicyclic sugar moiety, and the other comprises a 2′-substituted sugarmoiety. In certain embodiments, a gapmer has a sugar motif other thanE-K-K-(D)₉-K-K-E; E-E-E-E-K-(D)₉-E-E-E-E-E; E-K-K-K-(D)₉-K-K-K-E;K-E-E-K-(D)₉-K-E-E-K; K-D-D-K-(D)₉-K-D-D-K; K-E-K-E-K-(D)₉-K-E-K-E-K;K-D-K-D-K-(D)₉-K-D-K-D-K; E-K-E-K-(D)₉-K-E-K-E;E-E-E-E-E-K-(D)₈-E-E-E-E-E; or E-K-E-K-E-(D)₉-E-K-E-K-E, wherein K is anucleoside comprising a cEt sugar moiety and E is a nucleosidecomprising a 2′-MOE sugar moiety.

iii. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif, as described above for nucleoside motif. In suchembodiments, the internucleoside linkages in each of two wing regionsare different from the internucleoside linkages in the gap region. Incertain embodiments the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. The nucleoside motif is independently selected, sosuch oligonucleotides having a gapped internucleoside linkage motif mayor may not have a gapped nucleoside motif and if it does have a gappednucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

In certain embodiments, oligonucleotides comprise one or moremethylphosponate linkages. In certain embodiments, oligonucleotideshaving a gapmer nucleoside motif comprise a linkage motif comprising allphosphorothioate linkages except for one or two methylphosponatelinkages. In certain embodiments, one methylphosponate linkage is in thecentral gap of an oligonucleotide having a gapmer nucleoside motif.

iv. Certain Modification Motifs

Modification motifs define oligonucleotides by nucleoside motif (sugarmotif and nucleobase motif) and linkage motif. For example, certainoligonucleotides have the following modification motif:

A_(s)A_(s)A_(s)D_(s)D_(s)D_(s)D_(s)(^(N)D)_(s)D_(s)D_(s)D_(s)D_(s)B_(s)B_(s)B;

wherein each A is a modified nucleoside comprising a 2′-substitutedsugar moiety; each D is an unmodified 2′-deoxynucleoside; each B is amodified nucleoside comprising a bicyclic sugar moiety; ^(N)D is amodified nucleoside comprising a modified nucleobase; and s is aphosphorothioate internucleoside linkage. Thus, the sugar motif is agapmer motif. The nucleobase modification motif is a single modifiednucleobase at 8th nucleoside from the 5′-end. Combining the sugar motifand the nucleobase modification motif, the nucleoside motif is aninterrupted gapmer where the gap of the sugar modified gapmer isinterrupted by a nucleoside comprising a modified nucleobase. Thelinkage motif is uniform phosphorothioate. The following non-limitingTable further illustrates certain modification motifs:

TABLE 5 Certain Modification Motifs 5′-wing region Central gap region3′-wing region B_(S)B_(S)_(S)D_(S)D_(S)D_(S)D_(S)D_(S)D_(S)D_(S)D_(S)D_(S)A_(S)A_(S)A_(S)A_(S)A_(S)A_(S)A_(S)A AsBsBs DsDsDsDsDsDsDsDsDs BsBsAAsBsBs DsDsDsDs(ND)sDsDsDsDs BsBsA AsBsBs DsDsDsDsAsDsDsDsDs BsBsAAsBsBs DsDsDsDsBsDsDsDsDs BsBsA AsBsBs DsDsDsDsWsDsDsDsDs BsBsA AsBsBsBsDsDsDsDsDsDsDsDsDs BsBsAsBsB AsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsBBsBsAsBsBs DsDsDsDsDsDsDsDsDs BsBsA AsBsBs DsDsDsDsDsDsDsDsDsBsBsAsBsBsBsB AsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA AsAsAsBsAsAsDsDsDsDsDsDsDsDsDs BsBsA AsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsAAsAsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsAsA AsAsAsAsBsAsAsDsDsDsDsDsDsDsDsDs BsBsA AsBsAsBs DsDsDsDsDsDsDsDsDs BsAsBsA AsBsAsBsDsDsDsDsDsDsDsDsDs AsAsBsAsAs AsBsBs DsDsDsDsDsDsDsDsDs BsAsBsABsBsAsBsBsBsB DsDsDsDsDsDsDsDsDs BsAsBsA AsAsAsAsAs DsDsDsDsDsDsDsDsDsAsAsAsAsA AsAsAsAsAs DsDsDsDsDsDsDs AsAsAsAsA AsAsAsAsAsDsDsDsDsDsDsDsDsDs BsBsAsBsBsBsB AsAsAsBsBs DsDsDsDsDsDsDs BsBsAAsBsAsBs DsDsDsDsDsDsDsDs BsBsA AsBsAsBs DsDsDsDsDsDsDs AsAsAsBsBsAsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsA BsBs DsDsDsDsDsDsDsDs AsA AsAsDsDsDsDsDsDsDs AsAsAsAsAsAsAsA AsAsAs DsDsDsDsDsDsDs AsAsAsAsAsAsAAsAsAs DsDsDsDsDsDsDs AsAsAsAsAsA AsBs DsDsDsDsDsDsDs BsBsBsA AsBsBsBsDsDsDsDsDsDsDsDsDs BsA AsBs DsDsDsDsDsDsDsDsDs BsBsBsA AsAsAsBsBsDsDsDs(^(N)D)sDsDsDs BsBsAsAsA AsAsAsBsBs DsDsDsAsDsDsDs BsBsAsAsAAsAsAsBsBs DsDsDsBsDsDsDs BsBsAsAsA AsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsAAsAsBsBsBs DsDsDsDsDsDsDs BsBsBsAsA AsAsAsAsBs DsDsDsDsDsDsDs AsAsAsAsAsAsAsAsBsBs DsDsDsDsDsDsDs AsAsAsAsAs AsAsBsBsBs DsDsDsDsDsDsDsAsAsAsAsAs AsAsAsAsAs DsDsDsDsDsDsDs BsAsAsAsAs AsAsAsAsAsDsDsDsDsDsDsDs BsBsAsAsAs AsAsAsAsAs DsDsDsDsDsDsDs BsBsBsAsAs AsBsBsDsDsDsDs(^(N)D)s(^(N)D)sDsDsDs BsBsA AsBsBsDs(^(N)D)s(^(N)D)sDs(^(N)D)s(^(N)D)sDs- BsBsA (^(N)D)s(^(N)D)s AsBsBsDs(^(N)D)sDsDsDsDsDsDsDs BsBsA AsBsBs DsDs(^(N)D)sDsDsDsDsDsDs BsBsAAsBsBs Ds(^(N)D)s(^(N)D)sDsDsDsDsDsDs BsBsA AsBsBs DsDs(D)zDsDsDsDsDsDsBsBsA AsBsBs Ds(D)zDsDsDsDsDsDsDs BsBsA AsBsBs (D)zDsDsDsDsDsDsDsDsBsBsA AsBsBs DsDsAsDsDsDsDsDsDs BsBsA AsBsBs DsDsBsDsDsDsDsDsDs BsBsAAsBsBs AsDsDsDsDsDsDsDsDs BsBsA AsBsBs BsDsDsDsDsDsDsDsDs BsBsA AsBsAsBsDsDs(D)zDsDsDsDsDsDs BsBsBsAsAs AsAsAsBsBs DSDS(^(N)D)SDSDSDSDSDSDS ASAAsBsBsBs Ds(D)zDsDsDsDsDsDsDs AsAsAsBsBs AsBsBs DsDsDsDsDsDsDsDs(D)zBsBsA AsAsBsBsBs DsDsDsAsDsDsDs BsBsBsAsA AsAsBsBsBs DsDsDsBsDsDsDsBsBsBsAsA AsBsAsBs DsDsDsAsDsDsDs BsBsAsBsBsBsB AsBsBsBsDsDsDsDs(D)zDsDsDsDs BSA AsAsBsBsBs DsDsAsAsDsDsDs BsBsA AsBsBsDsDsDsDs(D)zDsDsDsDs BsBsBsA BsBs DsDs(^(N)D)sDs(^(N)D)sDsDsDsDsBsBsAsBsBsBsBwherein each A and B are nucleosides comprising differently modifiedsugar moieties, each D is a nucleoside comprising an unmodified 2′ deoxysugar moiety, each W is a modified nucleoside of either the first type,the second type or a third type, each ^(N)D is a modified nucleosidecomprising a modified nucleobase, s is a phosphorothioateinternucleoside linkage, and z is a non-phosphorothioate internucleosidelinkage.

In certain embodiments, each A comprises a modified sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moiety. Incertain embodiments, each A comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each A comprises a bicyclic sugar moiety. In certainembodiments, each A comprises a bicyclic sugar moiety selected fromamong cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certainembodiments, each A comprises a modified nucleobase. In certainembodiments, each A comprises a modified nucleobase selected from among2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certainembodiments, each B comprises a modified sugar moiety. In certainembodiments, each B comprises a 2′-substituted sugar moiety. In certainembodiments, each B comprises a 2′-substituted sugar moiety selectedfrom among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments,each B comprises a bicyclic sugar moiety. In certain embodiments, each Bcomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises amodified nucleobase. In certain embodiments, each B comprises a modifiednucleobase selected from among 2-thio-thymidine nucleoside and 5-propyneurindine nucleoside.

In certain embodiments, each W comprises a modified sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moiety. Incertain embodiments, each W comprises a 2′-substituted sugar moietyselected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certainembodiments, each W comprises a 5′-substituted sugar moiety. In certainembodiments, each W comprises a 5′-substituted sugar moiety selectedfrom among 5′-Me, and 5′-(R)-Me. In certain embodiments, each Wcomprises a bicyclic sugar moiety. In certain embodiments, each Wcomprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA,α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each W comprises asugar surrogate. In certain embodiments, each W comprises a sugarsurrogate selected from among HNA and F-HNA.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, and the other comprises a 2′-substituted sugar moiety. Incertain embodiments, one of A or B is an LNA nucleoside and the other ofA or B comprises a 2′-substituted sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside and the other of A or B comprises a2′-substituted sugar moiety. In certain embodiments, one of A or B is anα-L-LNA nucleoside and the other of A or B comprises a 2′-substitutedsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-MOE sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-MOE sugar moiety. In certain embodiments, one of A or Bis an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOEsugar moiety. In certain embodiments, one of A or B is an LNA nucleosideand the other of A or B comprises a 2′-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-F sugar moiety. In certain embodiments, one of A or B isan α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugarmoiety. In certain embodiments, one of A or B is an LNA nucleoside andthe other of A or B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, one of A or B is a cEt nucleoside and the other of A or Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A orB is an α-L-LNA nucleoside and the other of A or B comprises a2′-(ara)-F sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-substituted sugar moiety. In certain embodiments, A is anLNA nucleoside and B comprises a 2′-substituted sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-substitutedsugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-substituted sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-MOE sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-MOE sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-F sugar moiety. In certain embodiments,A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certainembodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugarmoiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and Bcomprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNAnucleoside and B comprises a 2′-(ara)-F sugar moiety. In certainembodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, A is an α-L-LNA nucleoside and Bcomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-MOE sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-F sugar moiety. In certain embodiments,B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certainembodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugarmoiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and Acomprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNAnucleoside and A comprises a 2′-(ara)-F sugar moiety. In certainembodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugarmoiety. In certain embodiments, B is an α-L-LNA nucleoside and Acomprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclicsugar moiety, another of A or B comprises a 2′-substituted sugar moietyand W comprises a modified nucleobase. In certain embodiments, one of Aor B is an LNA nucleoside, another of A or B comprises a 2′-substitutedsugar moiety, and W comprises a modified nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-substituted sugar moiety, and C comprises a modifiednucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-substituted sugar moiety,and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a modified nucleobase. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises amodified nucleobase. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a modified nucleobase. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a modified nucleobase. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-substituted sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a2′-substituted sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 2-thio-thymidine nucleobase. In certain embodiments, one ofA or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 2-thio-thymidine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a2-thio-thymidine nucleobase. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 2-thio-thymidine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and C comprises a 5-propyne uridine nucleobase. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, andC comprises a 5-propyne uridine nucleobase. In certain embodiments, oneof A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5-propyne uridinenucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5-propyne uridine nucleobase. In certain embodiments, one of A or B isan LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5-propyne uridine nucleobase. In certainembodiments, one of A or B is a cEt nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridinenucleobase. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is anα-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an LNA nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is a cEt nucleoside,another of A or B comprises a 2′-F sugar moiety, and W comprises a sugarsurrogate. In certain embodiments, one of A or B is an α-L-LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises asugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a sugar surrogate. In certain embodiments, one of A or B is acEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a sugar surrogate. In certain embodiments, one of A or Bis an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a HNA sugar surrogate. In certain embodiments, one of Aor B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a sugar HNA surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aHNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOEsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNAsugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a F-HNA sugar surrogate. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises aF-HNA sugar surrogate. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a F-HNA sugar surrogate. In certain embodiments, one of A or Bis a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a F-HNA sugar surrogate. In certain embodiments,one of A or B is an α-L-LNA nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-MeDNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety,and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one ofA or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-Fsugar moiety, and W comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNAnucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and Wcomprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A orB is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugarmoiety, and W comprises a 5′-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-MOE sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and Wcomprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of Aor B is a cEt nucleoside, another of A or B comprises a 2′-F sugarmoiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certainembodiments, one of A or B is an α-L-LNA nucleoside, another of A or Bcomprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugarmoiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety,another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is anLNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety,and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments,one of A or B is a cEt nucleoside, another of A or B comprises a2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.In certain embodiments, one of A or B is an α-L-LNA nucleoside, anotherof A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a5′-(R)-Me DNA sugar moiety.

In certain embodiments, at least two of A, B or W comprises a2′-substituted sugar moiety, and the other comprises a bicyclic sugarmoiety. In certain embodiments, at least two of A, B or W comprises abicyclic sugar moiety, and the other comprises a 2′-substituted sugarmoiety.

f. Certain Overall Lengths

In certain embodiments, the present invention provides oligomericcompounds including oligonucleotides of any of a variety of ranges oflengths. In certain embodiments, the invention provides oligomericcompounds or oligonucleotides consisting of X to Y linked nucleosides,where X represents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≦Y. For example, in certainembodiments, the invention provides oligomeric compounds which compriseoligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linkednucleosides. In embodiments where the number of nucleosides of anoligomeric compound or oligonucleotide is limited, whether to a range orto a specific number, the oligomeric compound or oligonucleotide may,nonetheless further comprise additional other substituents. For example,an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotideshaving 31 nucleosides, but, unless otherwise indicated, such anoligonucleotide may further comprise, for example one or moreconjugates, terminal groups, or other substituents. In certainembodiments, a gapmer oligonucleotide has any of the above lengths.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range.

g. Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention arecharacterized by their modification motif and overall length. In certainembodiments, such parameters are each independent of one another. Thus,unless otherwise indicated, each internucleoside linkage of anoligonucleotide having a gapmer sugar motif may be modified orunmodified and may or may not follow the gapmer modification pattern ofthe sugar modifications. For example, the internucleoside linkageswithin the wing regions of a sugar-gapmer may be the same or differentfrom one another and may be the same or different from theinternucleoside linkages of the gap region. Likewise, such sugar-gapmeroligonucleotides may comprise one or more modified nucleobaseindependent of the gapmer pattern of the sugar modifications. One ofskill in the art will appreciate that such motifs may be combined tocreate a variety of oligonucleotides. Herein if a description of anoligonucleotide or oligomeric compound is silent with respect to one ormore parameter, such parameter is not limited. Thus, an oligomericcompound described only as having a gapmer sugar motif without furtherdescription may have any length, internucleoside linkage motif, andnucleobase modification motif. Unless otherwise indicated, all chemicalmodifications are independent of nucleobase sequence.

h. Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachmentof one or more conjugate groups. In general, conjugate groups modify oneor more properties of the attached oligomeric compound including but notlimited to pharmacodynamics, pharmacokinetics, stability, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligomericcompound, such as an oligonucleotide. Conjugate groups includes withoutlimitation, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, thioethers, polyethers, cholesterols,thiocholesterols, cholic acid moieties, folate, lipids, phospholipids,biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine,fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groupshave been described previously, for example: cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached tooligonucleotides in oligomeric compounds. In certain embodiments,conjugate groups are attached to oligonucleotides by a conjugate linkinggroup. In certain such embodiments, conjugate linking groups, including,but not limited to, bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Conjugate linkinggroups are useful for attachment of conjugate groups, such as chemicalstabilizing groups, functional groups, reporter groups and other groupsto selective sites in a parent compound such as for example anoligomeric compound. In general a bifunctional linking moiety comprisesa hydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, theconjugate linker comprises a chain structure or an oligomer of repeatingunits such as ethylene glycol or amino acid units. Examples offunctional groups that are routinely used in a bifunctional linkingmoiety include, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties includepyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of anoligonucleotide (terminal conjugate groups) and/or at any internalposition.

In certain embodiments, conjugate groups are at the 3′-end of anoligonucleotide of an oligomeric compound. In certain embodiments,conjugate groups are near the 3′-end. In certain embodiments, conjugatesare attached at the 3′ end of an oligomeric compound, but before one ormore terminal group nucleosides. In certain embodiments, conjugategroups are placed within a terminal group. In certain embodiments, thepresent invention provides oligomeric compounds. In certain embodiments,oligomeric compounds comprise an oligonucleotide. In certainembodiments, an oligomeric compound comprises an oligonucleotide and oneor more conjugate and/or terminal groups. Such conjugate and/or terminalgroups may be added to oligonucleotides having any of the motifsdiscussed above. Thus, for example, an oligomeric compound comprising anoligonucleotide having region of alternating nucleosides may comprise aterminal group.

E. ANTISENSE COMPOUNDS

In certain embodiments, oligomeric compounds provided herein areantisense compounds. Such antisense compounds are capable of hybridizingto a target nucleic acid, resulting in at least one antisense activity.In certain embodiments, antisense compounds specifically hybridize toone or more target nucleic acid. In certain embodiments, a specificallyhybridizing antisense compound has a nucleobase sequence comprising aregion having sufficient complementarity to a target nucleic acid toallow hybridization and result in antisense activity and insufficientcomplementarity to any non-target so as to avoid non-specifichybridization to any non-target nucleic acid sequences under conditionsin which specific hybridization is desired (e.g., under physiologicalconditions for in vivo or therapeutic uses, and under conditions inwhich assays are performed in the case of in vitro assays).

In certain embodiments, the present invention provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

a. Certain Antisense Activities and Mechanisms

In certain antisense activities, hybridization of an antisense compoundresults in recruitment of a protein that cleaves of the target nucleicacid. For example, certain antisense compounds result in RNase Hmediated cleavage of target nucleic acid. RNase H is a cellularendonuclease that cleaves the RNA strand of an RNA:DNA duplex. The “DNA”in such an RNA:DNA duplex, need not be unmodified DNA. In certainembodiments, the invention provides antisense compounds that aresufficiently “DNA-like” to elicit RNase H activity. Such DNA-likeantisense compounds include, but are not limited to gapmers havingunmodified deoxyfuronose sugar moieties in the nucleosides of the gapand modified sugar moieties in the nucleosides of the wings.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid; a change in theratio of splice variants of a nucleic acid or protein; and/or aphenotypic change in a cell or animal.

In certain embodiments, compounds comprising oligonucleotides having agapmer nucleoside motif described herein have desirable propertiescompared to non-gapmer oligonucleotides or to gapmers having othermotifs. In certain circumstances, it is desirable to identify motifsresulting in a favorable combination of potent antisense activity andrelatively low toxicity. In certain embodiments, compounds of thepresent invention have a favorable therapeutic index (measure of potencydivided by measure of toxicity).

F. CERTAIN PHARMACEUTICAL COMPOSITIONS

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active antisense oligomericcompound.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present inventionto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present invention provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present invention provides methods ofadministering a pharmaceutical composition comprising an oligomericcompound of the present invention to an animal. Suitable administrationroutes include, but are not limited to, oral, rectal, transmucosal,intestinal, enteral, topical, suppository, through inhalation,intrathecal, intracerebroventricular, intraperitoneal, intranasal,intraocular, intratumoral, and parenteral (e.g., intravenous,intramuscular, intramedullary, and subcutaneous). In certainembodiments, pharmaceutical intrathecals are administered to achievelocal rather than systemic exposures. For example, pharmaceuticalcompositions may be injected directly in the area of desired effect(e.g., into the liver).

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligomeric compound having the nucleobase sequence “ATCGATCG”encompasses any oligomeric compounds having such nucleobase sequence,whether modified or unmodified, including, but not limited to, suchcompounds comprising RNA bases, such as those having sequence “AUCGAUCG”and those having some DNA bases and some RNA bases such as “AUCGATCG”and oligomeric compounds having other modified or naturally occurringbases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine basecomprising a methyl group at the 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the presentinvention and are not limiting. Moreover, where specific embodiments areprovided, the inventors have contemplated generic application of thosespecific embodiments. For example, disclosure of an oligonucleotidehaving a particular motif provides reasonable support for additionaloligonucleotides having the same or similar motif. And, for example,where a particular high-affinity modification appears at a particularposition, other high-affinity modifications at the same position areconsidered suitable, unless otherwise indicated.

Example 1 Synthesis of Oligomeric Compounds

The oligomeric compounds used in accordance with this disclosure may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as alkylatedderivatives and those having phosphorothioate linkages.

Oligomeric compounds: Unsubstituted and substituted phosphodiester (P═O)oligomeric compounds, including without limitation, oligonucleotides canbe synthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.

In certain embodiments, phosphorothioate internucleoside linkages (P═S)are synthesized similar to phosphodiester internucleoside linkages withthe following exceptions: thiation is effected by utilizing a 10% w/vsolution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forthe oxidation of the phosphite linkages. The thiation reaction step timeis increased to 180 sec and preceded by the normal capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (12-16 hr), the oligomeric compounds are recoveredby precipitating with greater than 3 volumes of ethanol from a 1 MNH₄OAc solution. Phosphinate internucleoside linkages can be prepared asdescribed in U.S. Pat. No. 5,508,270.

Alkyl phosphonate internucleoside linkages can be prepared as describedin U.S. Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate internucleoside linkages can beprepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050.

Phosphoramidite internucleoside linkages can be prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.

Alkylphosphonothioate internucleoside linkages can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,476,925.

Phosphotriester internucleoside linkages can be prepared as described inU.S. Pat. No. 5,023,243.

Borano phosphate internucleoside linkages can be prepared as describedin U.S. Pat. Nos. 5,130,302 and 5,177,198.

Oligomeric compounds having one or more non-phosphorus containinginternucleoside linkages including without limitationmethylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides,methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages can be prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal internucleoside linkages can be preparedas described in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide internucleoside linkages can be prepared as described inU.S. Pat. No. 5,223,618.

Example 2 Isolation and Purification of Oligomeric Compounds

After cleavage from the controlled pore glass solid support or othersupport medium and deblocking in concentrated ammonium hydroxide at 55°C. for 12-16 hours, the oligomeric compounds, including withoutlimitation oligonucleotides and oligonucleosides, are recovered byprecipitation out of 1 M NH₄OAc with >3 volumes of ethanol Synthesizedoligomeric compounds are analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresis.The relative amounts of phosphorothioate and phosphodiester linkagesobtained in the synthesis is determined by the ratio of correctmolecular weight relative to the −16 amu product (+/−32+/−48). For somestudies oligomeric compounds are purified by HPLC, as described byChiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtainedwith HPLC-purified material are generally similar to those obtained withnon-HPLC purified material.

Example 3 Synthesis of Oligomeric Compounds Using the 96 Well PlateFormat

Oligomeric compounds, including without limitation oligonucleotides, canbe synthesized via solid phase P(III) phosphoramidite chemistry on anautomated synthesizer capable of assembling 96 sequences simultaneouslyin a 96-well format. Phosphodiester internucleoside linkages areafforded by oxidation with aqueous iodine. Phosphorothioateinternucleoside linkages are generated by sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrousacetonitrile. Standard base-protected beta-cyanoethyl-diiso-propylphosphoramidites can be purchased from commercial vendors (e.g.PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway,N.J.). Non-standard nucleosides are synthesized as per standard orpatented methods and can be functionalized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligomeric compounds can be cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 4 Analysis of Oligomeric Compounds Using the 96-Well PlateFormat

The concentration of oligomeric compounds in each well can be assessedby dilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products can be evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates are diluted fromthe master plate using single and multi-channel robotic pipettors.Plates are judged to be acceptable if at least 85% of the oligomericcompounds on the plate are at least 85% full length.

Example 5 In Vitro Treatment of Cells with Oligomeric Compounds

The effect of oligomeric compounds on target nucleic acid expression istested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Cell linesderived from multiple tissues and species can be obtained from AmericanType Culture Collection (ATCC, Manassas, Va.).

The following cell type is provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays or RT-PCR.

b.END cells: The mouse brain endothelial cell line b.END was obtainedfrom Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany).b.END cells are routinely cultured in DMEM, high glucose (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells areroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells are seeded into 96-well plates(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a densityof approximately 3000 cells/well for uses including but not limited tooligomeric compound transfection experiments.

Experiments involving treatment of cells with oligomeric compounds:

When cells reach appropriate confluency, they are treated witholigomeric compounds using a transfection method as described.

LIPOFECTIN™

When cells reached 65-75% confluency, they are treated with one or moreoligomeric compounds. The oligomeric compound is mixed with LIPOFECTIN™Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reducedserum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achievethe desired concentration of the oligomeric compound(s) and aLIPOFECTIN™ concentration of 2.5 or 3 μg/mL per 100 nM oligomericcompound(s). This transfection mixture is incubated at room temperaturefor approximately 0.5 hours. For cells grown in 96-well plates, wellsare washed once with 100 μL OPTI-MEM™-1 and then treated with 130 μL ofthe transfection mixture. Cells grown in 24-well plates or otherstandard tissue culture plates are treated similarly, using appropriatevolumes of medium and oligomeric compound(s). Cells are treated and dataare obtained in duplicate or triplicate. After approximately 4-7 hoursof treatment at 37° C., the medium containing the transfection mixtureis replaced with fresh culture medium. Cells are harvested 16-24 hoursafter treatment with oligomeric compound(s).

Other suitable transfection reagents known in the art include, but arenot limited to, CYTOFECTIN™, LIPOFECTAMINE™, OLIGOFECTAMINE™, andFUGENE™. Other suitable transfection methods known in the art include,but are not limited to, electroporation.

Example 6 Real-Time Quantitative PCR Analysis of Target mRNA Levels

Quantitation of target mRNA levels is accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and coefficient of determination of the GAPDHand target signals generated from the multiplexed samples fall within10% of their corresponding values generated from the single-plexedsamples, the primer-probe set specific for that target is deemedmultiplexable. Other methods of PCR are also known in the art.

RT and PCR reagents are obtained from Invitrogen Life Technologies(Carlsbad, Calif.). RT, real-time PCR is carried out by adding 20 μL PCRcocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP,dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer,125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction iscarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol are carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by RT, real-time PCR are normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RIBOGREEN™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RIBOGREEN™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Example 7 Analysis of Oligonucleotide Inhibition of Target Expression

Antisense modulation of a target expression can be assayed in a varietyof ways known in the art. For example, a target mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently desired. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. One method of RNA analysis of the present disclosureis the use of total cellular RNA as described in other examples herein.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art. Methods for preparation ofpolyclonal antisera are taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9,John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 8 Design of Phenotypic Assays and In Vivo Studies for the Use ofTarget Inhibitors

Phenotypic Assays

Once target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Measurement of the expression of one or more of the genes of the cellafter treatment is also used as an indicator of the efficacy or potencyof the a target inhibitors. Hallmark genes, or those genes suspected tobe associated with a specific disease state, condition, or phenotype,are measured in both treated and untreated cells.

Example 9 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA is isolated according to Miura et al., (Clin. Chem., 1996,42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, theplate is gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate is transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates are incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plateis blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C., is added to each well, the plate is incubated on a90° C. hot plate for 5 minutes, and the eluate is then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA is isolated using an RNEASY 96™ kit and buffers purchased fromQiagen Inc. (Valencia, Calif.) following the manufacturer's recommendedprocedures. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 150μL Buffer RLT is added to each well and the plate vigorously agitatedfor 20 seconds. 150 μL of 70% ethanol is then added to each well and thecontents mixed by pipetting three times up and down. The samples arethen transferred to the RNEASY 96™ well plate attached to a QIAVAC™manifold fitted with a waste collection tray and attached to a vacuumsource. Vacuum is applied for 1 minute. 500 μL of Buffer RW1 is added toeach well of the RNEASY 96™ plate and incubated for 15 minutes and thevacuum is again applied for 1 minute. An additional 500 μL of Buffer RW1is added to each well of the RNEASY 96™ plate and the vacuum is appliedfor 2 minutes. 1 mL of Buffer RPE is then added to each well of theRNEASY 96™ plate and the vacuum applied for a period of 90 seconds. TheBuffer RPE wash is then repeated and the vacuum is applied for anadditional 3 minutes. The plate is then removed from the QIAVAC™manifold and blotted dry on paper towels. The plate is then re-attachedto the QIAVAC™ manifold fitted with a collection tube rack containing1.2 mL collection tubes. RNA is then eluted by pipetting 140 μL of RNAsefree water into each well, incubating 1 minute, and then applying thevacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 10 Target-Specific Primers and Probes

Probes and primers may be designed to hybridize to a target sequence,using published sequence information.

For example, for human PTEN, the following primer-probe set was designedusing published sequence information (GENBANK™ accession numberU92436.1, SEQ ID NO: 1).

Forward primer: AATGGCTAAGTGAAGATGACAATCAT (SEQ ID NO: 2)

Reverse primer: TGCACATATCATTACACCAGTTCGT (SEQ ID NO: 3) And the PCRprobe:

FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM isthe fluorescent dye and TAMRA is the quencher dye.

Example 11 Western Blot Analysis of Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 12 2-10-2 LNA Gapmers

The following gapmers comprising a 2-10-2 LNA motif were prepared usingthe procedures as described above. A subscript “1” indicates anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

TABLE 6 LNA Gapmers ISIS No. Motif Sequence Backbone SEQ. ID NO. 4578472-10-2 C₁C₁TGGTGTACACC₁C₁ Uniform PS 5 457848 2-10-2 G₁G₁TCCCTGCAGTA₁C₁Uniform PS 6

Example 13 FVII on-Target Knockdown

The inhibitory concentrations of ISIS No. 457847 and ISIS No. 457848 arepresented in Table 6. The inhibitory concentrations were calculated byplotting the doses of ISIS No. 457847 and ISIS No. 457848 versus thepercent inhibition of FVII mRNA expression achieved at eachconcentration, and noting the concentration of oligonucleotide at which10%, 20%, 50%, 80%, and 90% inhibition of FVII mRNA expression wasachieved compared to the control. This example demonstrates that bothIsis No. 457847 and Isis No. 457848 are potent inhibitors of FVII, andthat Isis No. 457848 is a more potent inhibitor of FVII than Isis No.457847. In this example, FVII is the “target,” all other genes are“off-target genes.”

TABLE 7 24 Dose (mg/kg) Dose (mg/kg) hours Isis No. 457847 Isis No.457848 IC₁₀ 39 21 IC₂₀ 30 15 IC₅₀ 19  9 IC₈₀ 12  5 IC₉₀  9  4

Example 14 In Vivo Acute Toxicity Study: Identification of SentinelGenes

Balb/c mice were subcutaneously administered saline, Isis No. 457847, or457848 at different doses as shown in the table below. Four out of theeight mice in each group were sacrificed 24 hours after administration.Immediately after each mouse was sacrificed, the livers were frozen inliquid nitrogen and then sent to Expression Analysis (Durham, N.C.) forwhole genome expression. Gene expression analysis on each of livers ofthe sacrificed mice was performed using a microarray to obtain wholegenome profiling.

Results from the microarray indicated that treatment with both Isis No.457847 and Isis No. 457848 induced more off-target down regulation thanoff-target up regulation.

For Isis No. 457848 at 100 mg/kg, it was found that 1617 off-targetgenes experienced a two-fold change in modulation of amount or activity,meaning that 1617 genes either decreased expression by at leasttwo-fold, or increased expression by at least two-fold. For Isis No.457847 at 200 mg/kg, it was found that 225 off-target genes experienceda two-fold change in modulation of amount or activity, meaning that 225genes either decreased expression by at least two-fold, or increasedexpression by at least two-fold.

Comparison and analysis of the changes in the modulation of amount oractivity of these off-target genes resulted in the identification of 143off-target genes (e.g. sentinel genes) that experienced a two-foldchange in modulation of amount or activity after administration of both100 mg/kg of Isis No. 457848 and 200 mg/kg of Isis No. 457847. These 143overlapping off-target genes are listed in Table 9.

TABLE 8 Dose Number Isis No. (mg/kg) of Mice Saline 0 8 457847 200 8457847 100 8 457847 50 8 457847 25 8 457847 12.5 8 Saline 0 8 457848 2008 457848 100 8 457848 50 8 457848 25 8 457848 12.5 8

TABLE 9 Off-Target Gene Identification Symbol Gene ID Rexo4 2276561810044A24Rik 76510 Atic 108147 Ccdc85b 240514 Capzb 12345 Abat 268860Pdss2 71365 Gcnt2 14538 Cadps2 320405 Vav2 22325 Prei4 74182 Prkag2108099 Dnajc12 30045 Rab8a 17274 Lrit1 239037 Pawr 114774 St3ga13 20441Pank1 75735 Ssbp3 72475 Cdo1 12583 Dusp8 18218 Kctd17 72844A530050D06Rik 104816 Fbxl17 50758 Zfhx3 11906 Ide 15925 1810020D17Rik66273 Msi2 76626 Pard3 93742 Ythdf3 229096 Usp18 24110 BC023892 2129434933407C03Rik 74440 Itpr1 16438 Dnaic1 68922 Tssc1 380752 BC048546232400 Ctnnbl1 66642 Luc712 192196 Snd1 56463 Ero11b 67475 Tyk2 54721Centg2 347722 Zfp260 26466 Zfp281 226442 Ptprk 19272 Ppp3ca 19055 Adam32353188 Ppp1r1b 19049 Crip2 68337 Ddc 13195 D630033011Rik 235302 Chn269993 BC018242 235044 Ergic1 67458 Mapkap1 227743 Wwox 80707 Stx8 55943Bcas3 192197 Exoc6b///Sec1512 75914 Ube2e2 218793 Parva 57342 Agpat267512 Adcy9 11515 Pkp4 227937 Pcbd2 72562 Fbxl20 72194 Scly 50880Macrod1 107227 Vti1a 53611 Abhd2 54608 4932417H02Rik 74370 Pgs1 74451Tmem162 76415 Adk 11534 BC029169 208659 Nedd41 83814 Ank 117321190005F20Rik 98685 Atg5 11793 Gck 103988 Mgmt 17314 Adam23 23792 Dym69190 Pitpnm2 19679 Nfib 18028 Bre 107976 Gphn 268566 Gapvd1 66691 Fars269955 Sfi1 78887 Tulp4 68842 Sds 231691 Sgms2 74442 Exoc4 20336 Pitpnc171795 Tox 252838 Lrba 80877 Npb 208990 LOC100046025 100046025 Myo1b17912 Ppm11 242083 Prnpip1 140546 Pdzrn3 55983 Atg7 74244 Supt3h 109115Hsd3b4 15495 Cryl1 68631 Ece1 230857 Mrap 77037 Smoc1 64075 Ext2 14043Ccdc91 67015 Hamp 84506 LOC100036521 100036521 Mnat1 17420 Eps1511 13859Alg14 66789 Paqr7 71904 Cdca7 66953 Arntl 11865 Slc17a2 2181032310009E04Rik 75578 Lace1 215951 BC057079 230393 F7 14068 1810026J23Rik69773 Uvrag 78610 Triobp 110253 Fto 26383 Herc2 15204 Parn 74108 Fndc3b72007 Sfxn5 94282 Epb4.1 269587 D930001I22Rik 228859 Immp21 93757Slc39a11 69806 Hamp2 66438 Rtp3 235636 6720458F09Rik 328162 Slc6a6 21366Dynll1 56455

Example 15 ALT and AST Toxicity

ALT and AST levels were measured in the remaining mice every 24 hours.All mice given Isis No. 457848 either were sacrificed after 48 hours ordied before the 48 hour time point. Any remaining mice were thensacrificed at 96 hours. ALT and AST levels were measured by taking asample of blood from each of the mice, centrifuging the sample, and thenanalyzing the plasma. ALT or AST levels greater than 10 times thebaseline indicated toxicity.

TABLE 10 ALT Levels at 24 hours Treatment ALT (IU/mL) Isis No. Dose(mg/kg) Duration (hours) Mean STDEV Saline 0 24 90 59 457847 100 24 4116 457847 200 24 35 2 457848 100 24 41 6 457848 200 24 73 42

TABLE 11 ALT Levels at 48 hours Treatment Dose Duration ALT (IU/mL) IsisNo. (mg/kg) (hours) Mean STDEV Saline  0 48   63  63 457847 100 48   30  0 457847 200 48   35   7 457848 100 48 17717 4243 457848 200 48 16667NA

TABLE 12 ALT Levels at 72 hours Treatment Dose Duration ALT (IU/mL) IsisNo. (mg/kg) (hours) Mean STDEV Saline  0 72  17  1 457847 100 72 178  64457847 200 72 284 180 457848 100 72 Lethal NA 457848 200 72 Lethal NA

TABLE 13 ALT Levels at 96 hours Treatment ALT (IU/mL) Isis No. Dose(mg/kg) Duration (hours) Mean STDEV Saline 0 96 24 4 457847 100 96 1632775 457847 200 96 15267 2620 457848 100 96 Lethal NA 457848 200 96Lethal NA

TABLE 14 AST Levels at 24 hours Treatment AST (IU/mL) Isis No. Dose(mg/kg) Duration (hours) Mean STDEV Saline 0 24 113 46 457847 100 24 8527 457847 200 24 75 13 457848 100 24 104 21 457848 200 24 91 35

TABLE 15 AST Levels at 48 hours Treatment AST (IU/mL) Isis No. Dose(mg/kg) Duration (hours) Mean STDEV Saline 0 48 116 157 457847 100 48 9837 457847 200 48 87 32 457848 100 48 16735 3426 457848 200 48 19859 NA

TABLE 16 AST Levels at 72 hours Treatment AST (IU/mL) Isis No. Dose(mg/kg) Duration (hours) Mean STDEV Saline 0 72 27 3 457847 100 72 15784 457847 200 72 164 65 457848 100 72 Lethal NA 457848 200 72 Lethal NA

TABLE 17 AST Levels at 96 hours Treatment AST (IU/mL) Isis No. Dose(mg/kg) Duration (hours) Mean STDEV Saline 0 96 41 3 457847 100 96 1026538 457847 200 96 9480 3094 457848 100 96 Lethal NA 457848 200 96 LethalNA

Example 16 Correlation Between Off-Target Gene Modulation and Toxicity:Overlapping Off-Target Genes

The degree of the change in modulation of amount or activity of each ofthe 143 overlapping off-target genes shown in Table 9 may be correlatedwith the amount of acute toxicity. For example, these off-target genesmay be correlated with the increase in AST or ALT levels described inExample 15. Identifying the off-target genes having the highestcorrelation between the degree of modulation of amount or activity ofexpression and acute toxicity would yield a sub-set of genes of interestfor further in-vitro validation.

Example 17 In Vitro Validation of Off-Target Genes and Identificationand Selection of Sentinel Genes

After identifying a sub-set of off-target genes of interest for furtherin-vitro validation, in vitro cells may be used to validate the sub-setof off-target genes, for example, in vitro cells may be used to validatethe 143 overlapping off-target genes shown in Table 9.

For example, to validate the 143 overlapping off-target genes shown inTable 9, primary hepatocytes from male Balb/c mice would be isolated.The isolated hepatocytes would be electroporated with water or Isis No.457848 or Isis No. 457847 at concentrations of 15 μM. At 2.5 hours afterelectroporation, the cells would then be refed with 100 μM of warmgrowth medium. At 16 hours after electroporation, the cells would bewashed and lysed with RLT+BME. The cells would then be shaken for 1minute before sealing and freesing at −80° C. Lysate would be used topurify the cells for RT-PCR analysis and genes would be measured byRT-PCR and Ribogreen and UV are read for each sample.

After obtaining the RT-PCR analysis of off-target genes thatdemonstrated strong amounts of modulation of amount or activity in vivo,the off-target genes that also show strong amounts of modulation ofamount or activity in vitro may now be identified. For example, if oneof the overlapping off-target genes shows a strong amount of downregulation in vivo upon the administration of a given oligonucleotide,and also demonstrates a strong amount of down regulation in vitro whenadministered the same oligonucleotide, then this off-target gene may beidentified as a good indicator of toxicity (e.g. sentinel gene). Now,one can administer a cell any number of different oligonucleotideshaving any number of motifs and modifications, and then monitor theregulation of the identified off-traget gene by RT-PCR or any othersuitable method known to those having skill in the art. In this mannerthe in vivo toxicity of any number of different oligonucleotides havingany number of motifs and modifications, may be predicted from an invitro assay.

Example 18 Correlation Between Off-Target Gene Modulation and Toxicity:Isis No. 457848

The degree of modulation of amount or activity each of the 1617off-target genes identified after administration of 100 mg/kg of IsisNo. 457848 may be correlated with the degree of increase in acutetoxicity. For example, these off-target genes may be correlated with anincrease in AST or ALT levels. Identifying the off-target genes havingthe highest correlation between modulation of amount or activity andacute toxicity would yield a sub-set of genes of interest for furtherin-vitro validation as detailed in Example 11 above.

Example 19 Correlation Between Off-Target Gene Modulation and Toxicity:Isis No. 457847

The degree of modulation of amount or activity of each of the 225off-target genes identified after administration of 200 mg/kg of IsisNo. 457847 may be correlated with the degree of increase in acutetoxicity. For example, these off-target genes may be correlated with anincrease in AST or ALT levels. Identifying the off-target genes havingthe highest correlation between modulation of amount or activity andacute toxicity would yield a sub-set of genes of interest for furtherin-vitro validation as detailed in Example 11 above.

Example 20 3-10-3 LNA Gapmers

The following 3-10-3 LNA gapmers were prepared using the procedures asdescribed above. A subscript “1” indicates a nucleoside comprising abicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. Each of thegapmers below have a full phosphorothioate backbone. Table 18 belowillustrates the sequences and targets of each compound.

TABLE 18 3-10-3 LNA Gapmers Isis No. Target Sequence SEQ ID NO. 569713NA/ASO ctrl G₁A₁C₁GCGCCTGAAGG₁T₁T₁ 7 571035 FVII C₁A₁G₁ATATAGGACTG₁G₁A₁8 571033 FXI A₁T₁C₁CAGAGATGCCT₁C₁C₁ 9 569714 FXI G₁G₁C₁CACCACGCTGT₁C₁A₁10 571034 FXI T₁G₁C₁CACCGTAGACA₁C₁G₁ 11 569715 SOD1G₁G₁A₁CACATTGGCCA₁C₁A₁ 12 569716 FVII C₁C₁C₁TGGTGTACACC₁C₁C₁ 13 569717PTEN A₁T₁C₁ATGGCTGCAGC₁T₁T₁ 14 569718 FVII T₁G₁G₁TCCCTGCAGTA₁C₁T₁ 15569719 FXI G₁T₁C₁TGTGCATCTCT₁C₁C₁ 16 569720 FXI G₁T₁C₁AGTATCCCAGT₁G₁T₁17 569721 SOD1 T₁G₁A₁GGTCCTGCACT₁G₁G₁ 18 554219 SurvivinC₁T₁C₁A₁ATCCATGGC₁A₁G₁C 19

Example 21 Off-Target Analysis of 3-10-3 LNA Gapmers

A series of antisense LNA containing oligonucleotides targeting a broadrange of targets were designed and synthesized as described above.Balb/c mice were separated into different groups and each group of micewas subcutaneously administered saline, or a single dose of Isis No.569713, 571035, 571033, 569714, 571034, 569715, 569716, 569717, 569718,569719, 569720, 569721, or 554219. In order to create a dose-responsecurve, the mice in each group were administered single doses of Isis No.569713, 571035, 571033, 569714, 571034, 569715, 569716, 569717, 569718,569719, 569720, 569721, and 554219 at different concentrations rangingfrom 1 mg/kg to 300 mg/kg. At 24 hours post administration, half of themice in each group for each dosage concentration were sacrificed.Immediately after each mouse was sacrificed, the livers were frozen inliquid nitrogen and then sent to Expression Analysis (Durham, N.C.) forwhole genome expression. Gene expression analysis on each of livers ofthe sacrificed mice was performed using a microarray to obtain wholegenome profiling. For each of the mice that were not sacrificed, sampleswere taken and ALT and AST levels were measured. Animals found deadprior to 96 hours were assigned an ALT value of 20000 IU/mL. Adose-response curve was then generated that plotted dose concentration(mg/kg) vs. ALT levels (IU/mL). The dose response curve for each of IsisNo. 569713, 571035, 571033, 569714, 571034, 569715, 569716, 569717,569718, 569719, 569720, 569721, or 554219 was then analyzed and used tocalculate the minimum dosage required to produce 1000 IU/ml ALT at 96 h.As the table below illustrates, doses ranging from 11 mg/kg to 300 mg/kgresulted in ALT levels greater than 1000 IU/ml for 7 compounds: IsisNos. 569716, 569717, 569718, 569719, 569720, 569721, and 554219. Theremaining compounds did not produce ALT levels greater than 1000 IU/ml,even after a single dose of 300 mg/kg.

TABLE 19 1^(st) Toxic Dose (mg/kg) > 1000 IU/ml ALT at 96 h 1^(st) ToxicDose On-Target Dose (mg/kg) > 1000 SEQ Isis No. Target Species (mg/kg)IU/ml ALT at 96 h ID NO. 569713 NA/ASO Mouse 300 >300 7 ctrl 571035 FVIIHuman 300 >300 8 571033 FXI Mouse 300 >300 9 569714 FXI Mouse 300 >30010 571034 FXI Mouse 300 >300 11 569715 SOD1 Mouse 300 >300 12 569716FVII Mouse 33 300 13 569717 PTEN Mouse <33 33 14 569718 FVII Mouse <33100 15 569719 FXI Mouse <11 11 16 569720 FXI Mouse 33 100 17 569721 SOD1Mouse <33 33 18 554219 Survivin Human 33 300 19

Example 22 Correlation Between Off-Target Gene Modulation and ALTIncrease

Gene expression analysis on each of the mice sacrificed at 24 hourspost-administration from Example 21 were analyzed. Expression of eachgene on the array was normalized to saline control. The fold change ofeach downregulated gene as measured at 24 hours post administration wasthen correlated to the increase in ALT measured at 96 hours. The genesthat illustrated the strongest correlation between down-regulation andan increase in ALT were then ranked according to r² values asillustrated in Table 20. Similarly, the genes that illustrated thestrongest correlation between up-regulation and an increase in ALT werethen ranked according to r² values as illustrated in Table 21.

TABLE 20 Down Regulated Genes Correlated to ALT Increase Gene RegulationEntrez (Up or Gene ID Gene Symbol r² Down) 74370 4932417H02Rik0.881570237 Down 75914 mKIAA0919///Sec1512///Exoc6b 0.877706718 Down50758 Fbx117 0.871834582 Down 69993 Chn2 0.868472413 Down 26383 Fto0.857328609 Down 666173 AK053274///mKIAA0532/// 0.852912584 DownVps13b///AK049111 80877 Lrba///Lba 0.831826949 Down 69955 Fars20.825377409 Down 217734 Pomt2 0.816819711 Down 211652 Wwc1 0.814841424Down 66795 Atg10 0.797656754 Down 14701 Gng12 0.793469536 Down 103677Smg6 0.789202811 Down 224008 2310008H04Rik 0.78792452  Down 19272 Ptprk0.785719243 Down 320405 Cadps2 0.785433781 Down 109115 Supt3h0.782544958 Down 20441 St3ga13 0.782160893 Down 74244 Atg7 0.771958613Down 75578 Fggy 0.770141201 Down 218793 Ube2e2 0.768562158 Down 93757Immp21 0.766370426 Down 192197 Bcas3 0.763707226 Down 17420 Mnat10.763237657 Down 16439 Itpr2 0.755316427 Down 11515 Adcy9 0.752314856Down 218103 S1c17a2 0.751397383 Down 27414 Sergef 0.74669734  Down 64075Smoc1 0.745821158 Down 69190 Dym 0.745739189 Down 18027 Nfia 0.745678305Down 23965 Odz3 0.745633647 Down 209224 Enox2 0.745318111 Down 72238Tbc1d5 0.74475067  Down 230393 BC057079 0.743701723 Down 12808 Cob10.743510516 Down 76626 Msi2 0.74312008  Down 13982 Esr1 0.743009136 Down58239 Dexi 0.741767493 Down 26936 AA536749 0.740805736 Down 13640 Efna50.738026555 Down 68975 Med27 0.737264649 Down 68916 Cdka11 0.73405334 Down 50771 Atp9b 0.73127855  Down 16010 Igfbp4 0.729708609 Down 20211Saa4 0.725536568 Down 72313 Fry1 0.723712037 Down 194401Mica13///Kiaa0819 0.722136142 Down 16438 Itpr1 0.721919362 Down 242083AK031097///Ppm11 0.720131701 Down 93742 Pard3 0.719281305 Down 17314Mgmt 0.717297987 Down 97287 Mtmr14 0.715716221 Down 18705 Pik3c2g0.711058186 Down 72007 Fndc3b 0.707287944 Down 12361 Cask 0.706570871Down 171212 Ga1nt10 0.704933641 Down 223754 Tbc1d22a 0.703695636 Down107227 Macrod1 0.698975352 Down 74374 C1ec16a 0.697481709 Down 208718Dis312 0.696060142 Down 74519 Cyp2j9 0.695058095 Down 268534 Sntg20.694530379 Down 81500 Si11 0.694446922 Down 219189 1300010F03Rik0.694202597 Down 13047 Cux1 0.69203827  Down 68618 1110012L19Rik0.688947418 Down 140546 Prnpip1 0.688766285 Down 20238 Atxn1 0.68713467 Down 71111 Gpr39 0.686579986 Down 14600 Ghr 0.683133879 Down 19266 Ptprd0.679746496 Down 74155 Errfi1 0.679613946 Down 227835 AK137808///Gtdc10.678056969 Down 320940 Atp11c 0.677044007 Down 108099 Prkag20.676318162 Down 239037 Lrit1 0.676002874 Down 213988 Tnrc6b 0.672130399Down 68178 Cgn11 0.67019584  Down 16795 Large 0.669312813 Down 268566Gphn 0.663682789 Down 319845 Bbs9 0.66198502  Down 18563 Pcx 0.659174176Down 94040 mKIAA1188///C1mn 0.659028801 Down 229487 Pet1121 0.658191741Down 78808 Stxbp5 0.65802987  Down 14043 Ext2 0.655674984 Down 94245Dtnbp1 0.653677345 Down 11881 Arsb 0.652843338 Down 224454 Zdhhc140.651947144 Down 105559 Mbn12 0.650606525 Down 13528 Dtnb 0.65026389 Down 19679 Pitpnm2 0.649808523 Down 15204 Herc2 0.649722071 Down 18606Enpp2 0.648732736 Down 53611 Vti1a 0.645315814 Down 238130Dock4///mKIAA0716 0.644365345 Down 99586 Dpyd 0.643599698 Down 74008Arsg 0.643248092 Down 110821 Pcca 0.642571153 Down 56463 Snd10.640221713 Down 67015 Ccdc91 0.637646731 Down 272428 Acsm5 0.636080214Down 14886 Gtf2i 0.635173991 Down 69806 S1c39a11 0.634795581 Down 110532Adarb1 0.632312769 Down 54604 Pcnx 0.631496126 Down 319885 Zcchc70.63146848  Down 102774 Bbs4 0.630868233 Down 243537 Uroc1 0.626857311Down 12558 Cdh2 0.626328157 Down 26396 Map2k2 0.626020226 Down 233977BC038349 0.625763342 Down 98496 5033414K04Rik 0.622537661 Down 269587Epb4.1 0.622028322 Down 330662 Dock1 0.621512901 Down 75735 Pank10.621009598 Down 54403 S1c4a4 0.61967098  Down 278279 Tmtc2 0.617639658Down 228730 Ncrna00153 0.617418242 Down 73652 BC099512 0.616894972 Down223254 Farp1 0.616836211 Down 18028 Nfib 0.616081199 Down 213498Arhgef11 0.615577511 Down 14718 Got1 0.614119517 Down 63955 Cab1es10.613107576 Down 68801 E1ov15 0.612792288 Down 74270 Usp20 0.612454854Down 17925 Myo9b 0.611954099 Down 83814 Nedd41///mKIAA0439 0.611375334Down 74088 0610012H03Rik 0.610067554 Down 233865 D430042O09Rik0.609934771 Down 216565 Ehbp1 0.609655313 Down 104718 Ttc7b 0.609371418Down 20338 Se111 0.608270142 Down 271564 Vps13a///CHAC 0.608063281 Down107986 Ddb2 0.606166164 Down 672511 Rnf213 0.605363852 Down 71602 Myo1e0.604467637 Down 17175 Masp2 0.603726298 Down 14585 Gfra1 0.603632842Down 15486 Hsd17b2 0.603323943 Down 192786 Rapgef6///mKIAA40520.60322277  Down 77987 Ascc3///AK144867 0.602976597 Down 18750 Prkca0.602949949 Down 57342 Parva 0.602253239 Down 14158 Fert2 0.601937675Down 29819 Stau2 0.601830334 Down 227743 Mapkap1 0.601738633 Down 241308AK140547///Ra1gps1 0.599029779 Down 20678 Sox5 0.59802968  Down 218865Chdh 0.597817574 Down 17127 Smad3 0.597594387 Down 54353 Skap20.597275862 Down 17120 Mad1///Mad111 0.597192128 Down 55983 Pdzrn30.596823197 Down 239985 Arid1b 0.596763305 Down 104816 Aspg 0.596526332Down 11749 Anxa6 0.59605341  Down 211673 Arfgef1 0.594411456 Down 50785Hs6st1 0.593828373 Down 71302 Arhgap26///mKIAA0621 0.593619374 Down104082 Wdr7 0.592931146 Down 100637 B230342M21Rik///N4bp211 0.592696965Down 65973 Asph 0.592305859 Down 544963 Iqgap2 0.591705376 Down 320011Ugcg11 0.5897969  Down 70661 BC033915 0.58976308  Down 215445mKIAA0665///Rab11fip3 0.589224941 Down 20679 Sox6 0.588615413 Down 76454Fbxo31 0.588503735 Down 68889 Ubac2 0.587940107 Down 94353 Hmgn30.586699681 Down 228602 4930402H24Rik 0.586564331 Down 108655 Foxp10.586468849 Down 171486 Cd9912 0.586443991 Down 223978C530044N13Rik///Cpped1 0.585623518 Down 76510 Trappc9///1810044A24Rik0.582060196 Down 29809 Rabgap11 0.581796202 Down 21372 Tb11x 0.581345739Down 23908 Hs2st1 0.581201637 Down 102566 Tmem16k///Ano10 0.579746255Down 347722 Agap1 0.579494425 Down 23938 Map2k5 0.579447394 Down 96935Susd4 0.578851694 Down 56878 Rbms1///AK011205 0.578650042 Down 100036521Gig18 0.578509922 Down 74440 4933407C03Rik///mKIAA1694 0.578167843 Down102644 Oaf 0.577524669 Down 54725 Cadm1 0.576998479 Down 22084 Tsc20.576392104 Down 56490 Zbtb20 0.575959654 Down 66253 Aig1 0.5755063 Down 246196 Zfp277///AK172713 0.575391946 Down 18201 Nsmaf 0.573842299Down 19045 Ppp1ca 0.573112804 Down 22325 Vav2 0.57267834  Down 23945Mg11 0.572572051 Down 26930 Ppnr 0.571883753 Down 76429 2310007H09Rik0.570953791 Down 231051 M113 0.57076196  Down 93834 Pe1i2 0.570020747Down 70834 Spag9///JSAP2 0.56678755  Down 12385 Ctnna1 0.566049233 Down20409 Ostf1 0.565624252 Down 52398 11-Sep 0.563024508 Down 17158 Man2a10.563022255 Down 18099 N1k 0.562409972 Down 216831 AU040829 0.561880909Down 11787 Apbb2 0.561732807 Down 68501 Nsmce2 0.561289726 Down 224671Btbd9 0.56054694  Down 229877 Rap1gds1 0.560281909 Down 68631 Cry110.560090087 Down 24059 S1co2a1 0.560080102 Down 22222 Ubr1 0.559857296Down 68732 Lac16a/Arrc16 0.559393676 Down 67074 Mon2 0.559331869 Down50754 Fbxw7 0.559122106 Down 19055 Ppp3ca 0.558920628 Down 107476AK040794///Acaca 0.558791978 Down 17155 Man1a 0.558773698 Down 207181Rbms3 0.558605596 Down 68465 Adipor2 0.558226759 Down 20192 Ryr30.557339048 Down 29807 Tpk1 0.557197421 Down 18624 Pepd 0.557093355 Down71764 C2cd21 0.555663167 Down 432442 Akap7 0.55457384  Down 103220BC030307 0.554449533 Down 105428 Fam149b 0.554372745 Down 20747 Spop0.554035756 Down 108138 Xrcc4 0.55386123  Down 208440 Dip2c 0.553415197Down 75472 1700009P17Rik 0.553150905 Down 72599 Pdia5 0.552830011 Down18534 Pck1 0.552604806 Down 68299 Vps53 0.552307087 Down 65967 Eefsec0.549508286 Down 68371 Pb1d 0.547859009 Down 227801 Dennd1a 0.547051646Down 17977 Ncoa1 0.545367828 Down 60344 Fign 0.54532852  Down

TABLE 21 Up Regulated Genes Correlated to ALT Increase Gene RegulationEntrez (Up or Gene ID Gene Symbol r² Down) 321000 4933421E11Rik0.828781401 Up 71989 Rpusd4 0.821214015 Up 68185 AK019895///Chchd80.812766903 Up 52477 Ange12 0.809563061 Up 14911 Thumpd3 0.804718994 Up69241 Po1r2d 0.80039532  Up 13197 Gadd45a 0.792167641 Up 107522 Ece20.784864986 Up 69549 2310009B15Rik 0.784498393 Up 68550 1110002N22Rik0.783538519 Up 233904 Setd1a 0.763731607 Up 69961 2810432D09Rik0.758340494 Up 66870 Serbp1 0.752501221 Up 67101 2310039H08Rik0.747968837 Up 270058 Mtap1s 0.744064198 Up 27260 P1ek2 0.741994766 Up69168 Bo1a1 0.738818242 Up 68115 AK172713///9430016H08Rik 0.738387466 Up73419 1700052N19Rik 0.738021033 Up 74132 Rnf6 0.734273477 Up 105663Thtpa 0.73343318  Up 227102 Ormd11 0.730356662 Up 243219 2900026A02Rik0.728731593 Up 20020 Po1r2a 0.727986948 Up 22629 Ywhah 0.727808243 Up16668 Krt18 0.727121927 Up 100515 Zfp518b 0.724764518 Up 66701 Spryd40.722478849 Up 104457 0610010K14Rik 0.717842951 Up 328099AU021838///Mipo11 0.715445754 Up 353188 Adam32 0.71430195  Up 699622810422O20Rik 0.713719025 Up 19039 Lga1s3bp 0.713151192 Up 353258 Ltv10.710511059 Up 68636 Fand1 0.709898912 Up 68327 0610007P22Rik0.709257388 Up 107701 Sf3b4 0.706098027 Up 218952 Fermt2 0.702510392 Up448850 Znhit3 0.702398266 Up 69228 Znf746 0.700863921 Up 71787 Trnau1ap0.700801498 Up 270106 Rp113 0.700293178 Up 68193 Rp124 0.699970694 Up18590 Pdgfa 0.699591372 Up 66664 Tmem41a 0.698860497 Up 208518 Cep780.698481328 Up 67781 I1f2 0.698448036 Up 70291 2510049J12Rik 0.69714475 Up 67489 Ap4b1 0.692430642 Up 76497 Ppp1r11 0.691954689 Up 77038 Arfgap20.690659625 Up 11676 A1doc 0.687782385 Up 15574 Hus1 0.687124907 Up51792 Ppp2r1a 0.686680263 Up 66083 Setd6 0.685885535 Up 22040AK036897///Trex1 0.685752435 Up 227522 Rpp38 0.685477194 Up 70223 Nars0.685365907 Up 28028 Mrp150 0.682327964 Up 17768 Mthfd2 0.682320691 Up69882 2010321M09Rik 0.682121395 Up 66606 Lrrc57 0.681908453 Up 231430Cox18 0.680319474 Up 22247 Umps 0.679307722 Up 11757 Prdx3 0.678891516Up 24110 Usp18 0.678408208 Up 16391 Isgf3g 0.677375454 Up 68979 No1110.676746807 Up 66653 Brf2 0.676339046 Up 67738 Ppid 0.676289037 Up 50918Myadm 0.674621176 Up 16691 Krt8 0.674433977 Up 69534 Avpi1 0.673529456Up 19340 Rab3d 0.670975146 Up 15374 Hn1 0.670724081 Up 70020 Ino80b0.670427391 Up 69573 2310016C08Rik 0.668340356 Up 66596 Gtf3a0.667461159 Up 83701 Srrt 0.666193892 Up 50887 Nsbp1 0.664511092 Up245841 Po1r2h 0.663503343 Up 68512 Tomm5 0.662237729 Up 55963 S1c1a40.661815979 Up 67832 Bxdc2 0.660961873 Up 276919 Gemin4 0.660309894 Up56716 Gb1 0.658651254 Up 100554 C87414///AA792892 0.658411355 Up 235302AK052711 0.657733584 Up 78394 Ddx52 0.656175645 Up 12238 Commd30.655418374 Up 108037 Shmt2 0.655013627 Up 69071 Tmem97 0.654795198 Up64406 Sp5 0.654602739 Up 68147 Gar1 0.654371413 Up 71988 Esco20.653785092 Up 66962 2310047B19Rik 0.653171224 Up 74097 Pop7 0.652820242Up 53317 P1rg1 0.650910996 Up 12464 Cct4 0.650849041 Up 20308 Cc190.650463831 Up 18950 Pnp1 0.647576204 Up 68145 Etaa1 0.646511359 Up76560 Prss8 0.645826963 Up 19671 Rce1 0.645558171 Up 216825 Usp220.644677729 Up 20174 Ruvb12 0.644207037 Up 23918 Impdh2 0.644160463 Up208990 Npb 0.643688534 Up 227715 Exosc2 0.643331854 Up 71916 Dus410.642261732 Up 69479 1700029J07Rik 0.641809029 Up 58248 1700123O20Rik0.641420014 Up 66401 Nudt2 0.640767939 Up 79554 G1tpd1 0.640567138 Up83703 Dbr1 0.64042371  Up 27356 Ins16 0.638467326 Up 20102 Rps4x0.6383911  Up 66658 Ccdc51 0.637337398 Up 69902 Mrto4 0.637181622 Up56209 Gde1 0.637166586 Up 71059 Hexim2 0.635654881 Up 234776 Atmin0.635066457 Up 74026 Ms11 0.63337818  Up 97541 Qars 0.632918392 Up225913 Dak 0.632596985 Up 105278 Ccrk 0.632551012 Up 76813 Armc60.632428466 Up 75616 2810008M24Rik 0.63153931  Up 75623Kde1c1///1700029F09Rik 0.631128792 Up 57357 Srd5a3 0.630154401 Up 233876Hirip3 0.629817864 Up 97159 A430005L14Rik 0.628500508 Up 230234 BC0265900.628443782 Up 12739 C1dn3///Wbscr25 0.628153864 Up 232337 Zfp6370.627445127 Up 14156 Fen1 0.62707061  Up 66248 A1g5 0.626283145 Up227154 A1s2cr2///Stradb 0.626171704 Up 622707 Rp129 0.625940105 Up 64295Tmub1 0.62580345  Up 26961 Rp18 0.624738153 Up 22666 Zfp161 0.62395442 Up 28010 D4Wsu114e 0.623435791 Up 71986 Ddx28 0.623316076 Up 18148 Npm10.622979601 Up 77286 Nkrf 0.622712352 Up 68002 1110058L19Rik 0.622440056Up 227644 Snapc4 0.622065994 Up 79059 Nme3 0.621781774 Up 226153 Peo10.621769908 Up 19921 Rp119 0.621648931 Up 18515 Pbx2 0.621321591 Up664968 2210411K11Rik 0.620986253 Up 67097 Rps10 0.62018374  Up 100040298Rps8 0.620012694 Up 230082 No16 0.619384089 Up 66481 Rps21 0.619315838Up 15495 Hsd3b4 0.619240278 Up 214424 Parp16 0.618433307 Up 18483 Pa1m0.617369158 Up 22051 Trip6 0.617293652 Up 217700 Acot6 0.617209221 Up68644 Abhd14a 0.61700942  Up 18100 Mrp140 0.616505506 Up 20042 Rps120.616251041 Up 217057 Ptrh2 0.615272427 Up 20821 Trim21 0.614943327 Up67602 Necap1 0.613081792 Up 231386 Ythdc1 0.612826851 Up 68080 Gpn30.612417706 Up 67996 Sfrs6 0.611610127 Up 27370ENSMUSG00000059775///Rps26 0.610651846 Up 69912 Nup43 0.610583513 Up19826 Rnps1 0.610497157 Up 101739 Psip1 0.609866248 Up 399566 Btbd60.609659323 Up 52626 Cdkn2aipn1 0.608900685 Up 19989 Rp17 0.607843346 Up13667 Eif2b4 0.607447824 Up 26441 Psma4 0.607302102 Up 22758 Zscan120.605678685 Up 667682 Rp131 0.605360844 Up 211255 Kbtbd7 0.605148748 Up69185 Dtwd1 0.605123393 Up 320226 4930473A06Rik///AK029637 0.604141033Up 216760 Mfap3 0.603674722 Up 67736 Ccdc130 0.603600403 Up 216150 Cdc340.603531354 Up 65972 Ifi30 0.603338123 Up 68044 Chac2 0.602240646 Up70240 Ufsp1 0.601764375 Up 67242 Gemin6 0.601630906 Up 16145 Igtp0.601376355 Up 56503 Ankrd49 0.600941885 Up 214489 AK206957///AK0506970.600265025 Up 269336 Ccdc32 0.600259909 Up 208595 ENSMUSG000000531780.6002454  Up 269955 Rccd1 0.600195992 Up 66172 Med11 0.600137828 Up100040353 2810416G20Rik 0.600052118 Up 14070 F8a 0.599086685 Up 66757Adat2 0.598703368 Up 20229 Sat1 0.598621988 Up 70650 Zcchc8 0.59793997 Up 52830 Pnrc2 0.597172683 Up 68366 Tmem129 0.596902461 Up 64655 Mrps220.596839038 Up 223626 4930572J05Rik 0.596704273 Up 269261 Rp1120.596680782 Up 225280 Ino80c 0.59649754  Up 66953 Cdca7 0.596271741 Up231915 Usp11 0.596199279 Up 208768 BC031781 0.595639474 Up 722752200002D01Rik 0.595226971 Up 192231 Hexim1 0.595082591 Up 208967 Thns110.594889153 Up 381792 AK009724 0.593903234 Up 77862 Thyn1///mThy280.593293926 Up 68879 Prpf6 0.592652691 Up 108098 Med21 0.592576963 Up22381 Wbp5 0.592072955 Up 105148 Iars 0.592040457 Up 68294 Mfsd100.591390672 Up 70021 Nt5dc2 0.590503402 Up 69861 2010003K11Rik0.590433914 Up 67676 Rpp21 0.589799041 Up 16205 Gimap1 0.588793908 Up66985 Rassf7 0.588743485 Up 217140 Scrn2 0.588506321 Up 70333 Cd3eap0.588454054 Up 240514 Ccdc85b 0.588420718 Up 109163 AK087382 0.588267639Up 56088 Psmg1 0.588101108 Up 108147 Atic 0.58752055  Up 67706 Tmem179b0.587035814 Up 67136 Kbtbd4 0.58667231  Up 212090 Tmem60 0.585540086 Up72655 2810026P18Rik 0.584433095 Up 449521 Zfp213 0.584222958 Up 107047Psmg2 0.582393668 Up 231841 AA881470 0.581273744 Up 66656 Eef1d0.581247585 Up 66170 Chchd5 0.581205624 Up 56791 Ube216 0.581062896 Up14865 Gstm4 0.58002253  Up 21339 Taf1a 0.579742839 Up 231583 S1c26a10.579592368 Up 57837 Era11 0.579409625 Up 27395 Mrp115///AK0178200.578731847 Up 69216 Ccdc23 0.578281126 Up 14113 Fb1 0.578077881 Up232236 C130022K22Rik 0.57804613  Up 554292 LOC554292 0.577954571 Up66973 Mrps18b 0.577310846 Up 66343 Tmem177 0.577302833 Up 59053 Brp160.577115872 Up 380712 T1cd2 0.576999831 Up 105014 Rdh14 0.576469122 Up226351 Tmem185b 0.575340901 Up 66489 Rp135 0.574558571 Up 66419 Mrp1110.574368522 Up 213541 Ythdf2 0.573980554 Up 18567 Pdcd2 0.573748231 Up26905 Eif2s3x 0.573638411 Up 11674 A1doa 0.573198881 Up 14534 Kat2a0.572866985 Up 66599 Rdm1 0.57158232  Up 67186 Rp1p2 0.571268544 Up219158 26103041G19Rik 0.571254833 Up 100043000 Rp13 0.570441285 Up 21924Tnnc1 0.569822051 Up 18648 Pgam1 0.569389772 Up 71726 Smug1 0.569262976Up 66358 2310004I24Rik 0.569017971 Up 60406 Sap30 0.568829534 Up 689491500012F01Rik 0.568756138 Up 101943 Sf3b3 0.568595763 Up 72536Tagap///Tagap1 0.568292844 Up 72388 Ripk4 0.568112975 Up 15931BC160215///Ids 0.568096931 Up 234309 Cbr4 0.568058926 Up 76800 Usp420.568758939 Up 22059 Trp53 0.566787444 Up 19175 Psmb6 0.565451493 Up213233 Tapbp1 0.565247118 Up 231872 Jtv1 0.5650143  Up 16549 Khsrp0.56470297  Up 231655 Oas11 0.564257429 Up 15239 Hgs 0.564155733 Up67427 Rps20 0.564113737 Up 15270 H2afx 0.563830152 Up 19172 Psmb40.563063414 Up 21816 Tgm1 0.562841486 Up 13163 Daxx 0.562685101 Up 24045C1k2//Scamp3 0.562455587 Up 225027 Sfrs7 0.562278505 Up 67843 S1c35a40.560934038 Up 214987 Chtf8 0.560908881 Up 23877 Fiz1 0.560848911 Up78372 Snrnp25 0.560175416 Up 52440 Tax1bp1 0.559550144 Up 53902 Rcan30.559014128 Up 69269 Scnm1 0.558513651 Up 12812 Coi1 0.558208066 Up97484 Cog8 0.557842373 Up 12567 Cdk4 0.557273744 Up 27756 Lsm20.557213698 Up 23849 K1f6 0.556951617 Up 12469 Cct8 0.556929134 Up 66910Tmem107 0.556716411 Up 57741 Noc21 0.556220338 Up 67211 Armc100.556028811 Up 97031 C430004E15Rik 0.555933123 Up 57785 Rangrf0.555885298 Up 210973 Kbtbd2 0.555784936 Up 16210 Impact 0.555775361 Up67390 Rnmt11 0.555625742 Up 14272 Fnta 0.555039858 Up 76650 Srxn10.554132183 Up 67053 Rpp14 0.554111651 Up 68763 AK003073 0.554047447 Up66480 Rp115 0.55308007  Up 382423 ENSMUSG00000074747 0.552900656 Up12366 Casp2 0.552565452 Up 101565 6330503K22Rik 0.552523353 Up 327959Xaf1 0.552462295 Up 56361 Pus1 0.552350536 Up 108660 Rnf187 0.552206839Up 56412 2610024G14Rik 0.551909024 Up 64656 Mrps23 0.551870129 Up 232087Mat2a 0.551664066 Up 217869 Eif5 0.551630047 Up 14155 Fem1b 0.551581421Up 19899 Rp118 0.550314468 Up 59054 Mrps30 0.550202135 Up 100039731Rp128 0.550102699 Up 107260 Otub1 0.549562967 Up 50772 Mapk6 0.549277905Up 21899 T1r6 0.548283895 Up 20088 Rps24 0.548272783 Up 13681 Eif4a10.548199142 Up 56176 Pigp 0.547310532 Up 104458 Rars 0.547261513 Up232491 Pyroxd1 0.546887964 Up 230721 Pabpc4 0.546883546 Up 20085 Rps190.546638269 Up 66242 Mrps16 0.54599052  Up 27407 Abcf2 0.5459486  Up80291 Ri1p12 0.545930544 Up 225160 Thoc1 0.545914264 Up 66614 Gpatch40.545621087 Up 100217418 AK009175 0.545345774 Up 217715 Eif2b20.545319888 Up

Example 23 Selection of Off-Target Genes as Sentinel Genes

Any off-target gene that demonstrates a correlation between upregulation or down regulation and an increase in ALT or some other valuepredictive of toxicity may be selected for in vitro validation. Incertain embodiments, a single gene that demonstrates correlation betweendown regulation and ALT increase may be selected for in vitrovalidation. In certain embodiments, a single gene that demonstratescorrelation between up regulation and ALT increase may be selected forin vitro validation. In certain embodiments, a gene from Table 20 thatdemonstrates correlation between down regulation and ALT increase may beselected for in vitro validation. In certain embodiments, a single genefrom Table 21 that demonstrates correlation between up regulation andALT increase may be selected for in vitro validation. In certainembodiments, one or more genes from Table 20 that demonstrates acorrelation between down regulation and ALT increase may be selected forin vitro validation. In certain embodiments, one or more genes fromTable 21 that demonstrates a correlation between up regulation and ALTincrease may be selected for in vitro validation. In certainembodiments, one or more genes from Table 20 and one or more genese fromTable 21 that demonstrate a correlation between modulation and ALTincrease may be selected for in vitro validation.

After identifying a sub-set of off-target genes, in vitro cells may beused to validate the sub-set of off-target genes. For example, in vitrocells may be used to validate the off-target genes shown in Table 20.For example, in vitro cells may be used to validate the off-target genesshown in Table 21.

To validate any of the off-target genes in Table 20 or Table 21, primaryhepatocytes from male Balb/c mice are isolated. The isolated hepatocytesare electroporated with water or any compound that produced an increasein ALT levels of greater than 1000 IU. At 2.5 hours afterelectroporation, the cells can then be refed with 100 μM of warm growthmedium. At 16 hours after electroporation, the cells are washed andlysed with RLT+BME. The cells are shaken for 1 minute before sealing andfreesing at −80° C. Lysate is used to purify the cells for RT-PCRanalysis. Genes may be measured by RT-PCR and Ribogreen and UV are readfor each sample.

After obtaining the RT-PCR analysis of off-target genes thatdemonstrated strong amounts of modulation of amount or activity in vivo,the off-target genes that also show strong amounts of modulation ofamount or activity in vitro may now be identified. For example, if oneof the off-target genes shows a strong amount of down regulation in vivoupon the administration of a given oligonucleotide, and alsodemonstrates a strong amount of down regulation in vitro whenadministered the same oligonucleotide, then this off-target gene may beidentified as a good indicator of toxicity (e.g. sentinel gene). In thefuture, one could then administer a cell any number of differentoligonucleotides having any number of motifs and modifications, and thenmonitor the regulation of the identified off-traget gene by RT-PCR orany other suitable method known to those having skill in the art. Inthis manner the in vivo toxicity of any number of differentoligonucleotides having any number of motifs and modifications, may beidentified.

Example 24 Median Length of mRNA Transcripts

Data from the whole genome expression in Example 21 was analyzed. Eachdown regulated gene was ranked according to its mRNA length. Eachunchanged gene was ranked according to its mRNA length. Each upregulated gene was ranked according to its mRNA length. The medianlength of each down regulated gene's mRNA, unchanged gene's mRNA, and upregulated gene's mRNA was then calculated. The results are presentedbelow in Table 22.

TABLE 22 Median Length of mRNA Transcripts Modulation Median Length DownRegulated 3962 Unchanged 2652 Up Regulated 1879

Example 25 Median Length of Pre-mRNA Transcripts

Data from the whole genome expression in Example 21 was analyzed. Eachdown regulated gene was ranked according to its pre-mRNA length. Eachunchanged gene was ranked according to its pre-mRNA length. Each upregulated gene was ranked according to its pre-mRNA length. The medianlength of each down regulated gene's pre-mRNA, unchanged gene'spre-mRNA, and up regulated gene's pre-mRNA was then calculated. Theresults are presented below in Table 23.

TABLE 23 Median Length of Pre-mRNA Transcripts Modulation Median LengthDown Regulated 176442 Unchanged 19862 Up Regulated 7673

Example 26 Combined Effects of Sentinel Genes

Six off-target genes, the modulation of which correlate strongly to ALTand/or AST increases were selected: RAPTOR, FTO, PPP3CA, PTPRK, IQGAP2,and ADK. These genes were identified as sentinel genes. Six 5-10-5 MOEgapmers with phosphorothioate backbones were then designed. Each 5-10-5MOE gapmer targeted a different sentinel gene. For example, the RAPTOR5-10-5 MOE gapmer would target and knock down the RAPTOR gene. Forexample, the FTO 5-10-5 MOE gapmer would target and knock down the FTOgene. For example, the PPP3CA 5-10-5 MOE gapmer would target and knockdown the PPP3CA gene. For example, the PTPRK 5-10-5 MOE gapmer wouldtarget and knock down the PTPRK gene. For example, the IQGAP2 5-10-5 MOEgapmer would target and knock down the IQGAP2 gene. For example, the ADK5-10-5 MOE gapmer would target and knock down the ADK gene. Balb/c micewere then separated into groups of 4 mice. Each group of mice was thengiven a subcutaneous 50 mg/kg dose seven times every other day of thevarious 5-10-5 MOE gapmers as illustrated in Table 24 below. Mice werethen bled at 24 hours after every other dose and a necropsy wasperformed 48 hours after the last dose. ALT was then measured. Isis No.:104838 is a 5-10-5 MOE gapmer that does not match a mouse target and wasused to ensure that the mice received standardized doses of gapmers.This example shows that the modulation of combinations of sentinel genesmay correlate to higher increases ALT levels as compared to increases inALT levels associated with the modulation of singular sentinel genes.

TABLE 24 Combined Effects of Sentinel Genes ASO (mg/kg) ALT (IU/mLRAPTOR FTO PPP3CA PTPRK IQGAP2 ADK 104838 Mean Std. Dev 0 0 0 0 0 0 027.6 12.5 0 0 0 0 0 0 200 77 8.8 0 0 0 0 0 0 300 131.8 20.5 0 0 0 0 0 500 40.5 10 0 0 0 0 50 0 0 50.8 8.7 0 0 0 50 0 0 150 103.3 11.2 0 0 50 0 00 150 44.3 8.7 0 0 50 50 50 50 100 201.5 23.7 0 50 0 0 0 0 150 46.5 8.10 50 0 50 50 50 100 199.3 82.8 0 50 50 0 50 50 100 127.5 28.8 0 50 50 500 50 100 179 101.1 0 50 50 50 50 0 100 210.5 32.4 0 50 50 50 50 50 50170.8 48.3 50 0 0 0 0 0 150 125.5 8.1 50 0 0 50 50 50 100 276.8 54.9 500 50 0 50 50 100 498.3 61 50 0 50 50 0 50 100 323.5 82 50 0 50 50 50 0100 247 47.5 50 0 50 50 50 50 50 300.5 109.8 50 50 0 0 50 50 100 546.5394 50 50 0 50 0 0 50 378.3 144.5 50 50 0 50 0 50 100 386.3 91.2 50 50 050 50 0 100 402.8 119.2 50 50 0 50 50 50 50 361.5 73.3 50 50 50 0 0 50100 354.8 95.3 50 50 50 0 50 0 100 553 178.7 50 50 50 0 50 50 50 851.532.3 50 50 50 50 0 0 100 785 286.3 50 50 50 50 0 0 0 929.3 100 50 50 5050 0 50 50 801.3 237.6 50 50 50 50 50 0 50 1169.3 257.8 50 50 50 50 5050 0 458.3 73.8

I claim:
 1. A method of predicting the in vivo toxicity of an oligomericcompound, wherein the method comprises: contacting a cell in vitro withthe oligomeric compound; and measuring the modulation of the amount oractivity of at least two sentinel genes, wherein the modulation of theamount or activity of the at least two sentinel genes correlates withtoxicity in vivo.
 2. The method of claim 1, wherein the oligomericcompound comprises a gapmer oligonucleotide consisting of 10 to 30linked nucleosides, wherein the gapmer oligonucleotide has a 5′ wingregion positioned at the 5′ end of a deoxynucleotide gap, and a 3′ wingregion positioned at the 3′ end of the deoxynucleotide gap.
 3. Themethod of claim 2, wherein each of the wing regions is between about 1to about 7 nucleotides in length.
 4. The method of claim 2, wherein eachof the wing regions is between about 1 to about 3 nucleotides in length.5. The method of claim 2, wherein the deoxy gap region is between about7 to about 18 nucleotides in length.
 6. The method of claim 2, whereinthe deoxy gap region is between about 11 to about 18 nucleotides inlength.
 7. The method of claim 2, wherein the deoxy gap region isbetween about 7 to about 10 nucleotides in length.
 8. The method ofclaim 2, wherein the oligomeric compound comprises at least one modifiednucleoside.
 9. The method of claim 8, wherein the modified nucleoside isa bicyclic modified nucleoside.
 10. The method of claim 8, wherein thebicyclic modified nucleoside is a locked nucleic acid (LNA) nucleoside.11. The method of claim 8, wherein the bicyclic modified nucleoside is aconstrained ethyl (cEt) nucleoside.
 12. The method of claim 8, whereinthe modified nucleoside is a 2′-modified nucleoside.
 13. The method ofclaim 12, wherein the 2′-modified nucleoside is substituted at the 2′position with a substituted or unsubstituted —O-alkyl or substituted orunsubstituted —O-(2-acetylamide), wherein the non-bicyclic 2′-modifiednucleoside comprises a 2′-OCH₃, 2′O(CH₂)₂OCH₃, or 2′-OCH₂C(O)—NR₁R₂,wherein R₁ and R₂ are independently hydrogen or substituted orunsubstituted alkyl or, in the alternative, are taken together to make aheterocyclic moiety.